Novel molecules of the card-related protein family and uses thereof

ABSTRACT

Novel CARD-12 polypeptides, proteins, and nucleic acid molecules are disclosed. In addition to isolated CARD-12 proteins, the invention further provides CARD-12, fusion proteins, antigenic peptides and anti-CARD-12 antibodies. The invention also provides CARD-12 nucleic acid molecules, recombinant expression vectors containing a nucleic acid molecule of the invention, host cells into which the expression vectors have been introduced and non-human transgenic animals in which a CARD-12 gene has been introduced or disrupted. Diagnostic, screening and therapeutic methods utilizing compositions of the invention are also provided.

RELATED APPLICATION INFORMATION

[0001] This application is a Continuation-in-part of application Ser.No. 09/697,089, filed Oct. 26, 2000, which claims priority fromprovisional application serial No. 60/161,822, filed Oct. 27, 1999. Theentire content of these applications is herein incorporated byreference.

BACKGROUND OF THE INVENTION

[0002] In multicellular organisms, homeostasis is maintained bybalancing the rate of cell proliferation against the rate of cell death.Cell proliferation is influenced by numerous growth factors and theexpression of proto-oncogenes, which typically encourage progressionthrough the cell cycle. In contrast, numerous events, including theexpression of tumor suppressor genes, can lead to an arrest of cellularproliferation.

[0003] In differentiated cells, a particular type of cell death calledapoptosis occurs when an internal suicide program is activated. Thisprogram can be initiated by a variety of external signals as well assignals that are generated within the cell in response to, for example,genetic damage. For many years, the magnitude of apoptotic cell deathwas not appreciated because the dying cells are quickly eliminated byphagocytes, without an inflammatory response.

[0004] The mechanisms that mediate apoptosis have been intensivelystudied. These mechanisms involve the activation of endogenousproteases, loss of mitochondrial function, and structural changes suchas disruption of the cytoskeleton, cell shrinkage, membrane blebbing,and nuclear condensation due to degradation of DNA. The various signalsthat trigger apoptosis are thought to bring about these events byconverging on a common cell death pathway that is regulated by theexpression of genes that are highly conserved from worms, such as C.elegans, to humans. In fact, invertebrate model systems have beeninvaluable tools in identifying and characterizing the genes thatcontrol apoptosis. Through the study of invertebrates and more evolvedanimals, numerous genes that are associated with cell death have beenidentified, but the way in which their products interact to execute theapoptotic program is poorly understood.

[0005] Caspases, a class of proteins central to the apoptotic program,are responsible for the degradation of cellular proteins that leads tothe morphological changes seen in cells undergoing apoptosis. Caspases(cysteinyl aspartate-specific proteinases) are cysteine proteases havingspecificity for aspartate at the substrate cleavage site. Generally,caspases are classified as either initiator caspases or effectorcaspases, both of which are zymogens that are activated by proteolysisthat generates an active species. An effector caspase is activated by aninitiator caspase which cleaves the effector caspase. Initiator caspasesare activated by an autoproteolytic mechanism that is often dependentupon oligomerization directed by association of the caspase with anadapter molecule.

[0006] Apoptotic signaling is dependent on protein-protein interactions.At least three different protein-protein interaction domains, the deathdomain, the death effector domain and the caspase recruitment domain(CARD), have been identified within proteins involved in apoptosis. Afourth protein-protein interaction domain, the death recruiting domain(DRD) was recently identified in murine FLASH (Imai et al. (1999) Nature398:777-85).

[0007] Caspases comprise a multi-gene family having at least 12 distinctfamily members (Nicholson (1999) Cell Death and Differentiation 6:1028).A relatively small fraction of cellular polypeptides (less than 200) arethought to serve as targets for cleavage by caspases. Because many ofthese caspase targets perform key cellular functions, their proteolysisis thought to account for the cellular and morphological events thatoccur during apoptosis. Members of the caspase gene family can bedivided by phylogenetic analysis into two major subfamilies, based upontheir relatedness to ICE (interleukin-1β converting enzyme; caspase-1)and CED-3. Alternate groupings of caspases can be made based upon theirsubstrate specificities.

[0008] Many caspases and proteins that interact with caspases possess aCARD domain. Hofmann et al. ((1997) TIBS 22:155) and others havepostulated that certain apoptotic proteins bind to each other via theirCARD domains and that different subtypes of CARD domains may conferbinding specificity, regulating the activity of various caspases, forexample.

[0009] Apoptosis in mammalian cells is mediated by large proteinfamilies that share sequence and structural similarity with the coreapoptotic proteins of Caenorhabditis elegans (Metzstein et al. (1998)Trends. Genet. 14:410). The nematode CED-4 protein and its human homologApaf-1 play central roles in apoptosis by transducing death signals tothe activation of caspases. Both CED-4 and Apaf-1 contain an N-terminalCARD domain that mediates caspase binding and a centrally locatednucleotide-binding site (NBS) domain. Unlike CED-4, Apaf-1 contains aC-terminal WD-40 domain that mediates protein activation in response tothe release of mitochondrial cytochrome c (Zou et al. (1997) Cell90:405; Li et al. (1997) Cell 91:479; Srinivasula et al. (1998) Mol.Cell. 1:949). Additional CED4/Apaf-1 family members include CARD-4, Nod2and CARD-7 (NAC/DEFCAP) (Bertin et al. (1999) J. Biol. Chem. 274:12955;Bertin et al. (2000) J. Biol. Chem. 275:41082; Inohara et al. (2000) J.Biol. Chem. 275:27823; Chu et al. (2001) J. Biol. Chem. 276:9239; Hlianget al. (2001) J. Biol. Chem. 276:9230). CARD4, Nod2 and CARD7 eachcontain NBS domains and effector CARD domains that mediate binding todownstream CARD-containing signaling partners. Both CARD-4 and Nod2assemble together with the CARD protein RICK and induce the activationof NF-kB. Recent evidence suggests that CARD-7 may play a role analogousto Apaf-1 and directly mediate caspase activation. In addition, eachprotein contains extensive leucine-rich repeats (LRR) that have beenproposed to function as binding sites for upstream regulators. Thestructure of CARD-4, Nod2 and CARD-7 is strikingly similar to plantNBS/LRR proteins that induce gene expression and cell death in responseto pathogen infection (Dixon et al. (2000) Proc. Nat'l. Acad. Sci. USA97:8807). Thus, CARD-4, Nod2 and CARD-7 likely play critical roles instress-activated signaling pathways and may be components of the hostinnate immune response.

SUMMARY OF THE INVENTION

[0010] The invention features nucleic acid molecules encoding humanCARD-12. CARD-12 has a CARD domain, a nucleotide binding site (NBS)domain, and a leucine rich repeat (LRR) domain. These domains are foundin a number of proteins that transmit signals that activate apoptoticand inflammatory pathways in response to stress and other stimuli. Uponactivation, CARD-12, like Apaf-1 (Zou et al. (1997) Cell 90:405-413),likely binds a nucleotide, allowing CARD-12 to bind to and activate aCARD-containing protein via a CARD-CARD interaction, leading tomodulation of apoptosis.

[0011] CARD-12 nucleic acids and polypeptides, as well as modulators ofCARD-12 activity or expression, are expected to be useful in themodulation of stress-related, apoptotic and inflammatory responses,e.g., for the treatment of apoptotic and inflammatory disorders. Inaddition, CARD-12 nucleic acids and polypeptides are expected to beuseful in the diagnosis of apoptotic and inflammatory disorders as wellas in screening assays which can be used to identify compounds which canbe used to modulate stress-related, apoptotic and inflammatoryresponses.

[0012] Many cytoplasmic plant proteins involved in response to plantpathogens, generally referred to as “R” proteins, have both an NBSdomain and an LRR domain (van der Bizen and Jones (1999) Current Biology8:226-228). R proteins are involved in both a rapid defense response(hypersensitive response) and more long-term nonspecific resistance(systemic acquired resistance). The hypersensitive response involvescell and tissue death that is localized to the site of infection. TheLRR domains of R proteins are believed to recognize and bind to pathogenproteins, triggering defensive responses. Many R proteins have an aminoterminal effector domain (e.g., a TIR domain or a leucine zipper domain)that is thought to play a role in downstream signaling of eventstriggered by infection and, possibly, other stresses.

[0013] The R proteins have some structural similarity to APAF-1, aprotein which mediates between Bcl-2, a negative regulator of apoptosis,and caspases. A domain, designated the NB-ARC domain(“nucleotide-binding adaptor shared by APAF-1, certain R gene productsand CED-4”) contains a series of motifs and residues that are conservedamong R proteins and APAF-1 (van der Bizen and Jones (1999) CurrentBiology 8:226-228). In addition to the NBS domain, APAF-1 has a CARDdomain, functionally analogous to the effector domain of R proteins, anda WD-40 domain, functionally analogous to the LRR domain of R proteins.

[0014] Similar to CARD-12, CARD-4 and CARD-7 have both an NBS domain andan LRR domain as well as a CARD domain (detailed information concerningCARD-4 and CARD-7 can be found in U.S. application Ser. No. 09/245,281,filed Feb. 5, 1999, U.S. application Ser. No. 09/207,359, filed Dec. 8,1998, U.S. application Ser. No. 09/099,041, filed Jun. 17, 1998, U.S.application Ser. No. 09/019,942, filed Feb. 6, 1998, and U.S.application Ser. No. 09/428,252, filed Oct. 27, 1999, all of which areincorporated herein by reference). The CARD domain, which is present ina number of apoptotic signaling molecules, is an effector domain thatthought to be involved in homophilic protein-protein interactions, e.g.,with downstream CARD-containing signaling molecules. For example, theCARD domain of CARD-4 interacts with the CARD domain of RICK (RIP2,CARDIAK), a serine-threonine kinase that activates NF-κB signalingpathways.

[0015] Other proteins structurally related to CARD-12 include NBS-1,Pyrin-1, PCD-1, PCD-2, and PCD-3.

[0016] NBS-1 has an NBS domain and a LRR domain, as well as a pyrindomain. Functionally analogous to the CARD domain of CARD-4, CARD-7, andCARD-12, the pyrin domain is an effector domain thought to be involvedin homophilic protein-protein interactions. Pyrin-1 also contains apyrin domain. Detailed information concerning NBS-1 and Pyrin-1 can befound in U.S. application Ser. No. 09/506,067, filed Feb. 17, 2000,which is incorporated herein by reference.

[0017] PCD-1, PCD-2, and PCD-3 each contain both an NBS domain and aleucine zipper domain. A leucine zipper domain, like the CARD domain andthe pyrin domain, is an effector domain thought to be involved inhomophilic protein-protein interactions. PCD-2 and PCD-3 also eachcontains LRR domains. PCD-1, which is truncated at is carboxy terminus,is also expected to contain an LRR domain. Detailed informationconcerning PCD-1, PCD-2, and PCD-3 can be found in U.S. application Ser.No. 09/563,876, filed May 3, 2000, which is incorporated herein byreference.

[0018] In general, an NBS domain includes a kinase 1 a domain (P-loop),and a kinase 2 domain (Walker B box). An LRR domain usually is composedof several leucine rich repeats.

[0019] Without being bound by a particular theory, it is possible thatCARD-12 participates in the network of interactions that lead to caspaseactivity. Human CARD-12 may play a functional role in caspase activationsimilar to that of Apaf-1 (Zou et al., Cell, 90:405-413, 1997). Forexample, upon activation, CARD-12 might bind a nucleotide, thus allowingCARD-12 to bind and activate a CARD-containing caspase via a CARD-CARDinteraction, leading to apoptotic death of the cell.

[0020] Accordingly, CARD-12 molecules are useful as modulating agents inregulating a variety of cellular processes including cell growth andcell death. In one aspect, this invention provides isolated nucleic acidmolecules encoding CARD-12 proteins or biologically active portionsthereof, as well as nucleic acid fragments suitable as primers orhybridization probes for the detection of CARD-12 encoding nucleicacids.

[0021] The invention encompasses methods of diagnosing and treatingpatients who are suffering from a disorder associated with an abnormallevel or rate (undesirably high or undesirably low) of apoptotic celldeath, abnormal activity of stress-related pathways of the endoplasmicreticulum (ER), abnormal activity of the Fas/APO-1 receptor complex,abnormal activity of the TNF receptor complex, or abnormal activity of acaspase by administering a compound that modulates the expression ofCARD-12 (at the DNA, mRNA or protein level, e.g., by altering mRNAsplicing) or by altering the activity of CARD-12. Examples of suchcompounds include small molecules, antisense nucleic acid molecules,ribozymes, and polypeptides.

[0022] Certain disorders are associated with an increased number ofsurviving cells, which are produced and continue to survive orproliferate when apoptosis is inhibited or occurs at an undesirably lowrate. CARD-12 and compounds that modulate the expression or activity ofCARD-12 can be used to treat or diagnose such disorders. These disordersinclude cancer (particularly follicular lymphomas, chronic myelogenousleukemia, melanoma, colon cancer, lung carcinoma, carcinomas associatedwith mutations in p53, and hormone-dependent tumors such as breastcancer, prostate cancer, and ovarian cancer). Such compounds can also beused to treat viral infections (such as those caused by herpesviruses,poxviruses, and adenoviruses). Failure to remove autoimmune cells thatarise during development or that develop as a result of somatic mutationduring an immune response can result in autoimmune disease. Thus, anautoimmune disorder can be caused by an undesirably low level ofapoptosis. Accordingly, CARD-12 and modulators of CARD-12 activity orexpression can be used to treat autoimmune disorders (e.g., systemiclupus erythematosis, immune-mediated glomerulonephritis, and arthritis).

[0023] Many diseases are associated with an undesirably high rate ofapoptosis. CARD-12 and modulators of CARD-12 expression or activity canbe used to treat or diagnose such disorders. A wide variety ofneurological diseases are characterized by the gradual loss of specificsets of neurons. Such disorders include Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis (ALS), retinitis pigmentosa,spinal muscular atrophy, Huntington's disease, and various forms ofcerebellar degeneration. The cell loss in these diseases does not inducean inflammatory response, and apoptosis appears to be the mechanism ofcell death. In addition, a number of hematologic diseases are associatedwith a decreased production of blood cells. These disorders includeanemia associated with chronic disease, aplastic anemia, chronicneutropenia, and the myelodysplastic syndromes. Disorders of blood cellproduction, such as myelodysplastic syndrome and some forms of aplasticanemia, are associated with increased apoptotic cell death within thebone marrow. These disorders could result from the activation of genesthat promote apoptosis, acquired deficiencies in stromal cells orhematopoietic survival factors, or the direct effects of toxins andmediators of immune responses. Two common disorders associated with celldeath are myocardial infarctions and stroke. In both disorders, cellswithin the central area of ischemia, which is produced in the event ofacute loss of blood flow, appear to die rapidly as a result of necrosis.However, outside the central ischemic zone, cells die over a moreprotracted time period and morphologically appear to die by apoptosis.Additional diseases associated with an undesirably high rate ofapoptosis include: ischemic and hypoxic brain injury, traumatic andexcitotoxic brain damage, neuronal transplantation, acute bacterialmeningitis, kidney ischemia/reperfusion injury, and liver disease.CARD-12 and modulators of CARD-12 may therefore be useful in treatingand diagnosing these conditions.

[0024] Populations of cells are often depleted in the event of viralinfection, with perhaps the most dramatic example being the celldepletion caused by the human immunodeficiency virus (HIV).Surprisingly, most T cells that die during HIV infections do not appearto be infected with HIV. Although a number of explanations have beenproposed, recent evidence suggests that stimulation of the CD4 receptorresults in the enhanced susceptibility of uninfected T cells to undergoapoptosis.

[0025] CARD-12 polypeptides, nucleic acids and modulators of CARD-12expression or activity can be used to treat inflammatory disorders andimmune system disorders. The inflammatory and immune disorders include,but are not limited to, chronic inflammatory diseases and disorders,such as Crohn's disease, reactive arthritis, including Lyme disease,insulin-dependent diabetes, organ-specific autoimmunity, includingmultiple sclerosis, Hashimoto's thyroiditis and Grave's disease, contactdermatitis, psoriasis, graft rejection, graft versus host disease,sarcoidosis, atopic conditions, such as asthma and allergy, includingallergic rhinitis, gastrointestinal allergies, including food allergies,eosinophilia, conjunctivitis, glomerular nephritis, certain pathogensusceptibilities such as helminthic (e.g., leishmaniasis), certain viralinfections, including HIV, and bacterial infections, includingtuberculosis and lepromatous leprosy.

[0026] Ischemia is often accompanied by inflammation that causes celldeath. Because CARD-12 is expected to play a role in stress-relatedresponse, inflammation and apoptosis, CARD-12 polypeptides, nucleicacids, and modulators of CARD-12 expression or activity can be used totreat cells death accompanying inflammatory responses triggered byischemia.

[0027] Invasive infection with Gram-negative bacteria and Gram-positivebacteria often results in septic shock. CARD-12 may recognize and bindcomponents of Gram-negative bacteria and Gram-positive bacteria or otherinfectious agents (e.g., intracellular parasites), triggering aninflammatory response. Thus, CARD-12 may play a role in innate immunesystem responses that is similar to that of Toll-like receptor 2 (TLR2),a receptor which has some structural similarity to plant R proteins andIL-1R. TLR2 is a signaling receptor that, in association with CD14, isactivated by LPS in a response that requires LPS-binding protein. Theinteraction of TLR2 with LPS leads to TLR2 oligomerization andrecruitment of IRAK (Yang et al. (1998) Nature 395:284-88; Yang et al(1999) J. Immunol. 163:639-43; and Yoshimura et al. (1999) J. Immunol.163:105). Thus, TLR2 is thought to be a direct mediator of signaling byLPS. TLR2 is also thought to mediate cell activation induced bypeptidoglycan and lipoteichoic acid, the main stimulatory components ofGram-positive bacteria (Schwandner et al. (1999) J. Biol. Chem.274:17406-09).

[0028] In addition to the aforementioned disorders, CARD-12polypeptides, nucleic acids, and modulators of CARD-12 expression oractivity can be used to treat septic shock and other disordersassociated with an innate immune response. For example, CARD-12 may bindto a component of an intracellular infectious agent or a component of aninfectious agent that is brought into a cell expressing CARD-12, e.g., acomponent that enters a cell through a receptor or is expressed by aviral gene.

[0029] In addition to the aforementioned disorders, CARD-12polypeptides, nucleic acids, and modulators of CARD-12 expression oractivity can be used to treat disorders of cell signaling and disordersof tissues in which CARD-12 is expressed.

[0030] The invention features a nucleic acid molecule which is at least45% (or 55%, 65%, 75%, 85%, 95%, or 98%) identical to the nucleotidesequence shown in SEQ ID NO: 1, SEQ ID NO:3, the nucleotide sequence ofthe cDNA insert of the plasmid deposited with the ATCC as AccessionNumber ______ (the “cDNA of ATCC ______”), or a complement thereof.

[0031] The invention features a nucleic acid molecule which includes afragment of at least 150 (300, 325, 350, 375, 400, 425, 450, 500, 550,600, 650, 700, 800, 900, 1000, 1300, 1600, 1900, 2100, 2400, 2700, 3000,or 3100) nucleotides of the nucleotide sequence shown in SEQ ID NO:1, orSEQ ID NO:3, or the nucleotide sequence of the cDNA ATCC ______, or acomplement thereof.

[0032] In an embodiment, a CARD-12 nucleic acid molecule has thenucleotide sequence shown in SEQ ID NO:1, or SEQ ID NO:3, or thenucleotide sequence of the cDNA of ATCC ______.

[0033] Also within the invention is a nucleic acid molecule whichencodes a fragment of a polypeptide having the amino acid sequence ofSEQ ID NO:2 or the polypeptide encoded by the cDNA of ATCC AccessionNumber ______. The fragment can comprise 15, 25, 50, 75, 100, 200, 300,400, 500, 600, 700, 800, 900, or 1000 contiguous amino acids of SEQ IDNO:2.

[0034] The invention includes a nucleic acid molecule which encodes anaturally occurring allelic variant of a polypeptide comprising theamino acid sequence of SEQ ID NO:2, wherein the nucleic acid moleculehybridizes to a nucleic acid molecule consisting of SEQ ID NO:1, SEQ IDNO:3, or the cDNA of ATCC under stringent conditions.

[0035] In general, an allelic variant of a gene will be readilyidentifiable as mapping to the same chromosomal location as the gene.

[0036] The invention also includes a nucleic acid molecule encoding anaturally occurring polypeptide, wherein the nucleic acid hybridizes toa nucleic acid molecule consisting of SEQ ID NO:3 under stringentconditions (e.g., hybridization in 6× sodium chloride/sodium citrate(SSC) at about 60° C., followed by one or more washes in 0.2× SSC, 0.1%SDS at 65° C.), and wherein the nucleic acid encodes a polypeptide of1020-1028 amino acids in length, preferably 1024 amino acids, having amolecular weight of about 116.1 kD prior to post-translationalmodifications. Thus, the invention encompasses a nucleic acid moleculewhich includes the sequence of the protein coding region of a naturallyoccurring mRNA (or the corresponding cDNA sequence) that is expressed ina human cell.

[0037] Also within the invention are: an isolated CARD-12 protein havingan amino acid sequence that is at least about 65%, preferably 75%, 85%,95%, or 98% identical to the amino acid sequence of SEQ ID NO:2; anisolated CARD-12 protein having an amino acid sequence that is at leastabout 85%, 95%, or 98% identical to the P-loop domain of SEQ ID NO:2(e.g., about amino acid residues 169 to 179 of SEQ ID NO:2); an isolatedCARD-12 protein having an amino acid sequence that is at least about85%, 95%, or 98% identical to the CARD domain of SEQ ID NO:2 (e.g.,about amino acid residues 1 to 88 of SEQ ID NO:2); an isolated CARD-12protein having an amino acid sequence that is at least about 65%,preferably 75%, 85%, 95%, or 98% identical to the nucleotide bindingsite core domain of SEQ ID NO:2 (e.g., about amino acid residues 161 to323 of SEQ ID NO:2); an isolated CARD-12 protein having an amino acidsequence that is at least about 65%, preferably 75%, 85%, 95%, or 98%identical to one or more of the leucine rich repeats of SEQ ID NO:2(e.g., about amino acids residues 762-789, 819-846, 847-874, and 938-965of SEQ ID NO:2); and an isolated CARD-12 protein having an amino acidsequence that is at least about 85%, 95%, or 98% identical to the NAIPhomology region of SEQ ID NO:2 (e.g., about amino acid residues 150 to1024 of SEQ ID NO:2).

[0038] Also within the invention are: an isolated CARD-12 protein whichis encoded by a nucleic acid molecule having a nucleotide sequence thatis at least about 65%, preferably 75%, 85%, or 95% identical to SEQ IDNO:3 or the cDNA of ATCC ______; an isolated CARD-12 protein which isencoded by a nucleic acid molecule having a nucleotide sequence at leastabout 85%, 95%, or 98% identical to the P-loop domain encoding portionof SEQ ID NO:3; an isolated CARD-12 protein which is encoded by anucleic acid molecule having a nucleotide sequence at least about 65%preferably 75%, 85%, or 95% identical the CARD domain encoding portionof SEQ ID NO:3; an isolated CARD-12 protein which is encoded by anucleic acid molecule having a nucleotide sequence at least about 65%preferably 75%, 85%, or 95% identical the NAIP homology encoding portionof SEQ ID NO:3; an isolated CARD-12 protein which is encoded by anucleic acid molecule having a nucleotide sequence at least about 65%preferably 75%, 85%, or 95% identical the nucleotide binding site domainencoding portion of SEQ ID NO:3; an isolated CARD-12 protein which isencoded by a nucleic acid molecule having a nucleotide sequence at leastabout 65% preferably 75%, 85%, or 95% identical to the LRR domainencoding portion of SEQ ID NO:3 or one or more leucine rich repeatencoding portions of SEQ ID NO:3; and an isolated CARD-12 protein whichis encoded by a nucleic acid molecule having a nucleotide sequence whichhybridizes under stringent hybridization conditions to a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:3 or the non-codingstrand of the cDNA of ATCC ______.

[0039] The CARD-12 nucleic acids, polypeptides, and antibodies of theinvention may be useful for mapping the location of the CARD-12 gene.

[0040] Another embodiment of the invention features CARD-12 nucleic acidmolecules which specifically detect CARD-12 nucleic acid molecules,relative to nucleic acid molecules encoding other members of the CARDsuperfamily and/or members of the NBS/LRR superfamily. For example, inone embodiment, a CARD-12 nucleic acid molecule hybridizes understringent conditions to a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or the cDNA of ATCC______, or a complement thereof. In another embodiment, the CARD-12nucleic acid molecule is at least 300 (350, 400, 450, 500, 550, 600,650, 700, 800, 900, 1000, 1300, 1600, 1900, 2100, 2400, 2700, 3000, or3100) nucleotides in length and hybridizes under stringent conditions toa nucleic acid molecule comprising the nucleotide sequence shown in SEQID NO:1, SEQ ID NO:3, the cDNA of ATCC ______, or a complement thereof.In another embodiment, an isolated CARD-12 nucleic acid moleculecomprises the CARD domain encoding portion of SEQ ID NO:3, or acomplement thereof. In yet another embodiment, the invention provides anisolated nucleic acid molecule which is antisense to the coding strandof a CARD-12 nucleic acid.

[0041] Another aspect of the invention provides a vector, e.g., arecombinant expression vector, comprising a CARD-12 nucleic acidmolecule of the invention. In another embodiment the invention providesa host cell containing such a vector. The invention also provides amethod for producing CARD-12 protein by culturing, in a suitable medium,a host cell of the invention containing a recombinant expression vectorsuch that a CARD-12 protein is produced.

[0042] Another aspect of this invention features isolated or recombinantCARD-12 proteins and polypeptides. Preferred CARD-12 proteins andpolypeptides possess at least one biological activity possessed bynaturally occurring human CARD-12, e.g., (1) the ability to formprotein:protein interactions with proteins in the apoptotic signalingpathway; (2) the ability to form CARD-CARD interactions with proteins inthe apoptotic signaling pathway; (3) the ability to bind a CARD-12ligand; and (4) the ability to bind to an intracellular target. Otheractivities include: (1) modulation of cellular proliferation; (2)modulation of cellular differentiation; (3) modulation of cellulardeath; (4) modulation of ER-specific apoptosis pathways; (5) modulationof the NF-kB pathway; (6) modulation of stress-responsive signalingpathways; and (7) modulation of an innate immune response.

[0043] The CARD-12 proteins of the present invention, or biologicallyactive portions thereof, can be operatively linked to a non-CARD-12polypeptide (e.g., heterologous amino acid sequences) to form CARD-12fusion proteins, respectively. The invention further features antibodiesthat specifically bind CARD-12 proteins, such as monoclonal orpolyclonal antibodies. In addition, the CARD-12 proteins or biologicallyactive portions thereof can be incorporated into pharmaceuticalcompositions, which optionally include pharmaceutically acceptablecarriers.

[0044] In another aspect, the present invention provides a method fordetecting the presence of CARD-12 activity or expression in a biologicalsample by contacting the biological sample with an agent capable ofdetecting an indicator of CARD-12 activity such that the presence ofCARD-12 activity is detected in the biological sample.

[0045] In another aspect, the invention provides a method for modulatingCARD-12 activity comprising contacting a cell with an agent thatmodulates (inhibits or stimulates) CARD-12 activity or expression suchthat CARD-12 activity or expression in the cell is modulated. In oneembodiment, the agent is an antibody that specifically binds to CARD-12protein. In another embodiment, the agent modulates expression ofCARD-12 by modulating transcription of a CARD-12 gene, splicing of aCARD-12 mRNA, or translation of a CARD-12 mRNA. In yet anotherembodiment, the agent is a nucleic acid molecule having a nucleotidesequence that is antisense to the coding strand of the CARD-12 mRNA orthe CARD-12 gene.

[0046] In one embodiment, the methods of the present invention are usedto treat a subject having a disorder characterized by aberrant CARD-12protein or nucleic acid expression or activity or related to CARD-12expression or activity by administering an agent which is a CARD-12modulator to the subject. In one embodiment, the CARD-12 modulator is aCARD-12 protein. In another embodiment the CARD-12 modulator is aCARD-12 nucleic acid molecule. In other embodiments, the CARD-12modulator is a peptide, peptidomimetic, or other small molecule.

[0047] The present invention also provides a diagnostic assay foridentifying the presence or absence of a genetic lesion or mutationcharacterized by at least one of: (i) aberrant modification or mutationof a gene encoding a CARD-12 protein; (ii) mis-regulation of a geneencoding a CARD-12 protein; (iii) aberrant RNA splicing; and (iv)aberrant post-translational modification of a CARD-12 protein, wherein awild-type form of the gene encodes a protein with a CARD-12 activity.

[0048] In another aspect, the invention provides a method foridentifying a compound that binds to or modulates the activity of aCARD-12 protein. In general, such methods entail measuring a biologicalactivity of a CARD-12 protein in the presence and absence of a testcompound and identifying those compounds which alter the activity of theCARD-12 protein.

[0049] The invention also features methods for identifying a compoundwhich modulates the expression of CARD-12 by measuring the expression ofCARD-12 in the presence and absence of a compound.

[0050] The invention also features methods for treating disordersassociated with inappropriate apoptosis (e.g., Alzheimer's diseases orother neurological disorders associated with neuronal apoptosis) bymodulating the expression or activity of CARD-12.

[0051] The invention also features methods for identifying a compoundthat alters (increases or decreases) the binding of CARD-12 (or a CARDdomain containing portion thereof) to a CARD domain containing protein(e.g., CARD-5) or a CARD domain containing portion thereof). The methodincludes measuring the binding of the protein (or polypeptides) to eachother in the presence and absence of a test compound and identifying thetest compound as a compound that alters binding if the binding in thepresence of test compound differs from the binding in the absence of thetest compound.

[0052] The invention also features a method for identifying a compoundthat binds to the NBS domain of CARD-12 by measuring the binding of atest compound to a polypeptide comprising the NBS domain of CARD-12. Thebinding can be measured in the presence of a nucleotide (e.g., an NTPsuch as ATP) for a competitive binding assay. Alternatively, the bindingcan be measured in the absence of a nucleotide that binds to the NBSsite.

[0053] The invention also features a method for identifying compoundsthat alter (increase or decrease) CARD-12 mediated apoptosis. Themethods include measuring apoptosis in the presence and absence of atest compound in cells expressing CARD-12 and in cells not expressingCARD-12 (or expressing less CARD-12). The CARD-12 expressed by the cellcan be encoded by a vector introduced into the cell. Thus, the cells canover-express CARD-12. A compound that alters apoptosis in the cellsexpressing CARD-12, but not in the cells not expressing CARD-12 (orexpressing less CARD-12), the compound is a candidate CARD-12 specificmodulator of apoptosis.

[0054] Compounds that increase the binding of CARD-12 to CARD-5 can beused to supplement chemotherapeutic agents.

[0055] Other features and advantages of the invention will be apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIGS. 1A-1E depict the cDNA sequence (SEQ ID NO:1) and predictedamino acid sequence (SEQ ID NO:2) of human CARD-12. The open readingframe of CARD-12 (SEQ ID NO:1) extends from nucleotide 36 to nucleotide3107 of SEQ ID NO:1 (SEQ ID NO:3).

[0057] FIGS. 2A-2E depict a predicted nucleotide sequence (SEQ ID NO:4)and predicted amino acid sequence (SEQ ID NO:5) of human CARD-12, basedupon the CARD-12 sequence derived from a genomic clone.

[0058]FIG. 3 depicts a hydropathy plot of CARD-12. Relativelyhydrophobic residues are above the dashed horizontal line, andrelatively hydrophilic residues are below the dashed horizontal line.The cysteine residues (cys) and N-glycosylation (Ngly) site areindicated by short vertical lines just below the hydropathy trace.

[0059]FIG. 4 depicts a plot showing the predicted structural features ofCARD-12. This figure shows the predicted alpha regions (Garnier-Robsonand Chou-Fasman), the predicted beta regions (Gamier-Robson andChou-Fasman), the predicted turn regions (Gamier-Robson and Chou-Fasman)and the predicted coil regions (Garnier-Robson). Also included in thefigure is a hydrophilicity plot (Kyte-Doolittle), the predicted alphaand beta-amphipathic regions (Eisenberg), the predicted flexible regions(Karplus-Schulz), the predicted antigenic index (Jameson-Wolf) and thepredicted surface probability plot (Emini).

[0060]FIG. 5A depicts an alignment of amino acids 2-88 of human CARD-12(amino acid residues 2-88 of SEQ ID NO:2) with a CARD domain (SEQ IDNO:7) derived from a hidden Markov model.

[0061]FIG. 5B depicts an alignment of amino acids 764-791 of humanCARD-12 (amino acid residues 764-791 of SEQ ID NO:2) with a consensusleucine rich repeat (SEQ ID NO:8) derived from a hidden Markov model.

[0062]FIG. 5C depicts an alignment of amino acids 821-848 of humanCARD-12 (amino acid residues 821-848 of SEQ ID NO:2) with a consensusleucine rich repeat (SEQ ID NO:8) derived from a hidden Markov model.

[0063]FIG. 5D depicts an alignment of amino acids 849-872 of humanCARD-12 (amino acid residues 849-872 of SEQ ID NO:2) with a consensusleucine rich repeat (SEQ ID NO:8) derived from a hidden Markov model.

[0064]FIG. 5E depicts an alignment of amino acids 938-965 of humanCARD-12 (amino acid residues 938-965 of SEQ ID NO:2) with a consensusleucine rich repeat (SEQ ID NO:8) derived from a hidden Markov model.

[0065] FIGS. 6A-6C depict an alignment of amino acids 150-1024 of humanCARD-12 (amino acid residues 150-1024 of SEQ ID NO:2) with amino acids451-1232 of neuronal AIP (SEQ ID NO:9).

[0066]FIG. 7 depicts an alignment of the CARD domain of human CARD-12(amino acids 1-88 of SEQ ID NO:2) with the CARD domains of CARD-4,CARD-7, and Apaf-1.

[0067]FIG. 8 depicts schematic drawings of the domain structures ofhuman CARD-12, human CARD-4, human CARD-7, human Nod2, and Apaf-1.

[0068]FIG. 9 depicts the results of a mammalian two-hybrid assay used toidentify CARD domains that interact with the CARD domain of CARD-12.

[0069]FIG. 10 depicts the results of co-immunopreciptation analysis.Panel A: After 24 hrs, extracts were prepared and immunoprecipitated(IP) with a monoclonal antibody to the T7 epitope. Theimmunoprecipitates were analyzed by SDS-PAGE and immunoblotted with ananti-myc polyclonal antibody. Panel B: The cellular extracts were alsoimmunoblotted (WB) with anti-T7 antibody. Panel C: Extracts were alsoimmunoprecipitated with a monoclonal antibody to myc, followed by WBwith an anti-myc polyclonal antibody.

DETAILED DESCRIPTION OF THE INVENTION

[0070] The present invention is based, in part, on the identification ofa cDNA sequence encoding a human CARD-12 protein. A nucleotide sequenceencoding a human CARD-12 protein is shown in FIGS. 1A-1E (SEQ ID NO:1;SEQ ID NO:3 includes the open reading frame only). A predicted aminoacid sequence of CARD-12 protein is also shown in FIGS. 1A-1E (SEQ IDNO:2).

EXAMPLE 1 Identification and Characterization of CARD-12

[0071] A BAC clone (GenBank™ Accession Number AL121653) was searched toidentify potential exons. The predicted protein sequences were thensearched using a profile of known CARD domains. This search led to theidentification of a sequence predicted to encode a CARDdomain-containing protein later identified as CARD-12. FIGS. 2A-2Edepict a nucleotide sequence (SEQ ID NO:4) assembled from the BAC clonewhich includes a predicted open reading frame (SEQ ID NO:6; nucleotides1-3612 of SEQ ID NO:4) encoding a 1204 amino acid protein (SEQ ID NO:5).

[0072] The CARD-12 sequence assembled from the BAC clone was used tofurther characterize the CARD-12 cDNA sequence. A search of aproprietary electronic cDNA database (created from a human lymphocytelibrary) using sequences derived from the 3′ end of SEQ ID NO:4 led tothe identification of a cDNA containing the 3′ end of the CARD-12 cDNA.The sequence of the 5′ end of the CARD-12 cDNA was determined by asearch of the Incyte (Palo Alto, Calif.) Life Gold Templates cDNAelectronic database using sequences from the 5′ end of SEQ ID NO:4. AcDNA sequence was identified (Incyte clone number 328193) that contained366 nucleotides of the 5′ portion of the CARD-12 cDNA. Primers weredesigned corresponding to the 5′ untranslated region and the 3′ carboxyterminus coding region of CARD-12. PCR amplification using these primerswas performed on a placenta cDNA library. The sequencing of PCR productsled to the identification of the cDNA sequence of CARD-12 (SEQ ID NO:1).

[0073] The CARD-12 cDNA sequence (SEQ ID NO:1) has some differencescompared to that predicted by the assembly of the predicted CARD-12exons of the BAC clone (SEQ ID NO:4). These differences are generallylocated at the 5′ and 3′ end of the cDNA. Where the CARD-12 sequencesdepicted in SEQ ID NO:1 and SEQ ID NO:4 differ, SEQ ID NO:1 correspondsto the CARD-12 cDNA sequence.

[0074] FIGS. 1A-1E depict the sequence of a 3133 nucleotide cDNA (SEQ IDNO:1) which includes a predicted open reading frame (SEQ ID NO:3;nucleotides 36-3107 of SEQ ID NO:1) encoding a 1024 amino acid humanCARD-12 protein (SEQ ID NO:2). Human CARD-12 is predicted to be anintracellular protein.

[0075] The predicted amino acid sequence of human CARD-12 was comparedto amino acid sequences of known proteins and various motifs wereidentified. The 1024 amino acid human CARD-12 protein includes twoN-glycosylation sites (e.g., about amino acid residues 539-542 and764-767 of SEQ ID NO:2); a glycosaminoglycan attachment site (e.g.,about amino acid residues 171-174 of SEQ ID NO:2); four cAMP- andcGMP-dependent protein kinase phosphorylation site (e.g., about aminoacid residues 60-63, 228-231, 453-456, and 985-988 of SEQ ID NO:2); 11protein kinase C phosphorylation sites (e.g., about amino acid residues72-74, 171-173, 188-190, 226-228, 403-405, 536-538, 566-568, 665-667,689-691, 710-712, and 973-975 of SEQ ID NO:2); 20 casein kinase IIphosphorylation sites (e.g., about amino acid residues 72-75, 94-97,133-136, 215-218, 279-282, 365-368, 415-418, 445-448, 460-463, 479-482,497-500, 541-544, 553-556, 607-610, 665-668, 725-728, 742-745, 851-854,920-923, and 973-976 of SEQ ID NO:2); a tyrosine kinase phosphorylationsite (e.g., about amino acid residues 71-78 of SEQ ID NO:2); 15N-myristoylation sites (e.g., about amino acid residues 62-67, 156-161,187-192, 211-216, 291-296, 380-385, 516-521, 618-623, 699-704, 754-759,760-765, 894-899, 928-933, 946-951, and 959-964 of SEQ ID NO:2); anamidation site (e.g., about amino acid residues 451-454 of SEQ ID NO:2);and an ATP/GTP-binding site motif A (P-loop) (e.g., about amino acidresidues 169-179 of SEQ ID NO:2).

[0076]FIG. 3 depicts a hydropathy plot of CARD-12. Relativelyhydrophobic residues are above the dashed horizontal line, andrelatively hydrophilic residues are below the dashed horizontal line.The cysteine residues (cys) and N-glycosylation (Ngly) site areindicated by short vertical lines just below the hydropathy trace.

[0077] A plot showing the predicted structural features of CARD-12 ispresented in FIG. 4. This figure shows the predicted alpha regions(Garnier-Robson and Chou-Fasman), the predicted beta regions(Gamier-Robson and Chou-Fasman), the predicted turn regions(Gamier-Robson and Chou-Fasman) and the predicted coil regions(Garnier-Robson). Also included in the figure is a hydrophilicity plot(Kyte-Doolittle), the predicted alpha and beta-amphipathic regions(Eisenberg), the predicted flexible regions (Karplus-Schulz), thepredicted antigenic index (Jameson-Wolf) and the predicted surfaceprobability plot (Emini).

[0078] An analysis of the predicted CARD-12 amino acid sequence showedit to contain a CARD domain (e.g., about amino acid residues 1-88 of SEQID NO:2), a nucleotide binding site (NBS) domain (e.g., about amino acidresidues 161-323 of SEQ ID NO:2), and three leucine rich repeats (LRR;e.g., about amino acid residues 762-789, 819-846, 847-874, and 938-965of SEQ ID NO:2) which form a LRR domain (e.g., about amino acid residues762-965 of SEQ ID NO:2). Within the predicted NBS domain there is akinase 1a domain (P-loop) (e.g., about amino acid residues 169-179 ofSEQ ID NO:2) and a kinase 2 domains (e.g., about amino acid residues245-248 of SEQ ID NO:2).

[0079]FIG. 5A depicts an alignment of amino acids 2-88 of human CARD-12(amino acid residues 2-88 of SEQ ID NO:2) with a consensus CARD domain(SEQ ID NO:7) derived from a HMM.

[0080] FIGS. 5B-5E each depict an alignment of one of the four leucinerich repeats within the LRR domain of CARD-12 (amino acid residues764-791 of SEQ ID NO:2 (FIG. 5B), amino acid residues 821-848 of SEQ IDNO:2 (FIG. 5C), amino acid residues 849-872 of SEQ ID NO:2 (FIG. 5D),and amino acid residues 938-965 of SEQ ID NO:2 (FIG. 5E)) with aconsensus LRR (SEQ ID NO:8) derived from a HMM.

[0081] The domain alignments depicted in FIGS. 5A-5E were identified byhomology searching using consensus domains derived from hidden Markovmodels (HMMs). HMMs can be used to perform multiple sequence alignmentand very sensitive database searching, using statistical descriptions ofa domain's consensus sequence. For more information on HMM searches,see, e.g., http://hmmer.wustl.edu/. In the alignments of FIGS. 5A-5E asingle letter amino acid designation at a position on the line betweenthe CARD-12 sequence and the HMM-generated consensus domain sequenceindicates an exact match between the two. A “+” in this middle lineindicates a conservative substitution at the particular residue ofCARD-12. Amino acid residues located in the domains identified by theHMM search may be important for the appropriate functioning of theCARD-12 protein. For this reason, amino acid substitutions with respectto the sequence of SEQ ID NO:2 that are outside of the domainshomologous to HMM consensus domains may be less detrimental to theactivity of the CARD-12 protein.

[0082] The C-terminal portion of CARD-12 (amino acids 150 to 1024 of SEQID NO:2) bears some similarity to the N-terminus of neuronal AIP(GenBank™ Accession Number Q13075; Roy et al. (1995) Cell 80:167-178).FIGS. 6A-6C depict an alignment of amino acids 451-1232 of neuronal AIP(SEQ ID NO:9) and amino acids 150-1024 of human CARD-12.

EXAMPLE 2 Additional Characterization of CARD-12 Domains

[0083] CARD-12 includes seven NACHT (NAIP, CIIA, HET-E and TP1) NTPasedomains. The seven NACHT NTPase domains are at amino acids 169-186 ofSEQ ID NO:2 (P-Loop/Walker A Box /Motif I); amino acids 196-220 of SEQID NO:2 (Walker B Box/Mg⁺⁺ binding domain Motif II); amino acids 229-253of SEQ ID NO:2 (Motif III); amino acids 261-282 of SEQ ID NO:2 (MotifIV); amino acids 330-351 of SEQ ID NO:2 (Motif V); amino acids 414-430of SEQ ID NO:2 (Motif VI) and amino acids 438-457 of SEQ ID NO:2 (MotifVII). Other members of the NACHT NTPase family include: CARD-4, CARD-7,and NAIP (Koonin et al. (2001) Trends Biochem. Sci. 25:223).

[0084] Additional analysis of the leucine-rich repeat domain of CARD-12revealed that the domain extends from amino acid 656-1021 of SEQ ID NO:2and contains 13 leucine-rich repeats (amino acids 656-686, 687-708,711-737, 738-761, 762-788, 789-817, 819-845, 846-874, 875-901, 903-930,936-962, 965-993, and 994-1021 of SEQ ID NO:2.

[0085] Additional analysis of CARD-12 revealed that the nucleotidebinding site domain extends from amino acid 169 to amino acid 456 of SEQID NO:2.

[0086] As discussed above, the CARD domain of CARD-12 extends from aminoacid 1-88 of SEQ ID NO:2. FIG. 7 depicts an alignment of the CARD domainof human CARD-12 (amino acids 1-88 of SEQ ID NO:2) with the CARD domainsof CARD-4, CARD-7, and Apaf-1.

[0087] As discussed above, CARD-12 includes domains present in membersof the CED/Apaf-1 family. FIG. 8 depicts schematic drawings of thedomain structures of human CARD-12 as well as human CARD-4, CARD-7,Nod2, and Apaf-1, all of which are members of the CED/Apaf-1 family.

[0088] Molecules that binding to and alter the activity of the NBSdomain of CARD-12 may be useful for modulating the activity of CARD-12.For example, molecules can be tested for their ability to modulate,e.g., antagonize, the hydrolysis of an NTP, e.g., ATP, by thenucleotide-binding site of CARD-12. Methods of detecting the hydrolysisof ATP by a nucleotide-binding site are described in, for example,Gadsby et al. (1999) Physiol. Rev. 79:S77-S107. Additional assays thatmight be used are described in Li et al., (1996) J. Biol. Chem. 271:28463-28468.

EXAMPLE 3 Expression Analysis

[0089] Northern blot analysis of CARD-12 expression using a human adulttissue blot (Clontech, La Jolla, Calif.) revealed that a 3.3-kilobaseCARD-12 transcript is present in human lymphoid tissues, includingspleen, peripheral blood lymphocytes, bone morrow and fetal liver.

[0090] An affinity purified polyclonal CARD-12 antibody was used toinvestigate expression in primary normal human epithelial cells(Epipanel; Clonetics, Inc.). Affinity-purified CARD-12 antibody wasraised in rabbits injected with the 15-mer peptide LWRQESLQSVKNTTEcorresponding to residues 527-542 of CARD-12 (Research Genetics). Theanalysis revealed that a about 120 kD CARD-12 protein is expressed inhuman renal cortical primary epithelial cells, human mammary primaryepithelial cells, human renal proximal tubule primary epithelial cells,human bronchial primary epithelial cells, and human prostate primaryepithelial cells. Expression was also detected in human primaryepithelial cells, and muscle primary cells.

EXAMPLE 4 CARD-12 Mediates Apoptosis

[0091] To determine a possible role for CARD-12 in apoptosis signaling arecombinant adenovirus expressing full length CARD-12 was constructed.Briefly, CARD-12 was expressed using a dual-adenovirus based,tetracycline-regulatable expression system. A similar system haspreviously been shown to work extremely efficiently both in vitro and invivo. CARD-12 was cloned into the adenovirus transfer vector pLE11f,placing the CARD-12 gene under the transcriptional control of thetetracycline-regulatable promoter. An internal downstream ribosome entrysite allowed a modified green fluorescent protein (KGFP) to be expressedoff the same transcript. E1/E3-deleted adenovirus was then generated byhomologous recombination in 911 cells (Fallaux et al. (1996) Hum. GeneTher. 7:215), plaque-purified and protein expression verified by Westernblot. VERO cells were transfected at an MOI of about 20. As a control,cells were similarly transfected with an adenovirus vector expressingKGFP only. VERO cells were plated in 96 well dishes and transfected thefollowing day with adenovirus (MOI of about 20). Cells were fixed (4%paraformaldehyde in 0.15M PBS) 36 hours after transfection. The nucleiwere then stained with Hoescht 3342 and the percentage of apoptoticversus healthy nuclei in transfected cells was scored. Within 36 hoursof transfection with CARD-12 expressing adenovirus, the transfectedcells were undergoing apoptosis, as indicated by rounding up andmembrane blebbing. Additionally, 45.2±4.0% (mean ±SE) ofCARD-12-transfected cells had condensed, pyknotic nuclei, whereas only3.8±1.1% of KGFP-transfected cells showed signs of apoptosis.Adenovirus-mediated transfection of either CARD-12 or KGFP in VERO cellsresulted in at least 90% of cells transfected. Thus, CARD-12 is thenovel member of the Apaf-1/CED4 protein family (along with CARD-4,CARD-7, Nod2 and Apaf-1) shown to activate downstream cell deathsignals. As a member of the Apaf-1/CED4 family, transduction of aproapoptotic signal through CARD-12 is likely to be mediated byCARD/CARD interaction with one or more CARD-containing signalingmolecules.

EXAMPLE 5 CARD-12 Interacts with CARD-5 (ACS)

[0092] A mammalian two-hybrid analysis was used to identify potentialbinding partners of CARD-12. In this analysis the binding of theN-terminal CARD of CARD-12 to the CARD domains of 23 known proteins wasassessed. The analysis involved the used of a plasmid,pCMV-CARD-12-CARD/AD, constructed by inserting the CARD domain ofCARD-12 (residues 1-83) into pCMV-AD (Stratagene; La Jolla, Calif.). Inthe assay, 293T cells in 6-well plates (35-mm wells) were transfectedwith the following plasmids: 750 ng of pCMV-CARD-12/AD, 750 ng ofpCMV-BD fused to individual CARD domains, 250 ng of pFR-Luc fireflyreporter (Stratagene), and 250 ng of pRL-TK renilla reporter (Promega).Cells were harvested 24 hrs after transfection, and firefly luciferaseactivity was determined using the Dual-Luciferase Reporter Assay System(Promega). In addition, renilla luciferase activity was determined andused to normalize transfection efficiencies. The results of thetwo-hybrid analysis are shown in FIG. 9. The CARD of CARD-12 interactedwith the CARD of CARD-5, resulting in an 10-fold increase in relativeluciferase activity. The CARD domain of CARD-12 also had a strongself-association, resulting in a 30-fold increase in relative luciferaseactivity (FIG. 9). Co-expression of CARD-12 CARD with other CARD domainsfailed to activate luciferase expression indicating that the CARD ofCARD-12 interacts selectively with the CARD of CARD-5, a protein thatmediates apoptosis induced by chemotherapeutic agents (Masumoto et al.(1999) J. Biol. Chem. 274:33835).

EXAMPLE 6 The Pyrin Domain of CARD-5 Mediates Apoptosis

[0093] CARD-5, which consists of an N-terminal PYRIN domain and aC-terminal CARD domain is a proapoptotic protein and is subject tomethylation-induced silencing in a number of breast cancers. This,latter observation suggests that CARD-5 may play a fumdamental role incell death. Given that CARD-12 and CARD-5 interact via their respectiveCARDs, the PYRIN domain of CARD-5 may function as its proapoptoticeffector domain. To examine this possibility, adenovirus vectorsexpressing CARD-5 truncation mutants were created. Briefly, CARD-5truncation mutants containing the PYRIN domain or the CARD domain, werecloned into the adenovirus transfer vector pLE11f, placing the gene ofinterest under the transcriptional control of thetetracycline-regulatable promoter. An internal ribosome entry sitedownstream to the gene of interest allows a modified KGFP to beexpressed off the same transcript. E1/E3-deleted adenovirus was thengenerated by homologous recombination in 911 cells, plaque-purified andprotein expression verified by Western blot. VERO cells were transfected(MOI=20) with recombinant adenovirus expressing full length CARD-5(AdTRE-CARD51-195), or either of the PYRIN domain(AdTRE-CARD-5-PYR1-150) or CARD (AdTRE-CARD-5-CARD74-195) of CARD-5alone. Thirty-six hours after transfection cells were fixed and stainedwith the nuclear dye Hoescht 33342 and the percentage of apoptoticversus healthy nuclei in transfected cells was then scored. Western blotfor the FLAG epitope-tag indicate relative levels of expression fromeach vector. Thirty-six hours after transfection of VERO cells with anadenovirus expressing full length CARD-5 60.4±1.6% of cells wereundergoing apoptosis. Interestingly, transfection with the PYRIN domainof CARD-5 alone resulted 66.1±5.4% cell death. Expression of the CARD ofCARD-5 alone resulted in virtually no cell death (2.6±0.6%). Theseresults suggest that a PYRIN domain can play a functional role inapoptosis signaling, and substantiates the emerging hypothesis thatPYRIN-containing proteins represent another important family of proteinsinvolved in transducing the complex signals of apoptosis.

EXAMPLE 7 CARD-12 Interacts with Caspase-1

[0094] An additional mechanism by which CARD-12 may cause cell death isby the activation of upstream caspases via a CARD/CARD interaction. Theproform of caspase-1 and caspase-9 both contain an N-terminal CARD.Therefore, caspase-1 and caspase-9 were investigated as possible CARD-12signaling partners following transient overexpression in cells. Briefly,293T cells transfected with plasmids were lysed in 50 mM Tris, pH 8.0,120 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40 buffer and incubated witheither a T7 (Sigma) or myc monoclonal antibody (SantaCruz Biotechnology,Inc.). The immune complexes were precipitated with protein G-Sepharose(Amersham Pharmacia Bio), washed extensively, and then subjected toSDS-polyacrylamide gel electrophoresis and immunoblotted with polyclonalantibody to myc (SantaCruz). The results of the co-immunoprecipitationanalysis are shown in FIG. 10. Immunoprecipitation of T7-taggedcaspase-1, but not T7-tagged procaspase-9, co-precipitated myc-taggedCARD-12 (FIG. 10, Panel A, lanes 1 and 3, respectively). These resultsindicate a possible role for CARD-12 in caspase-1 signaling. Thestructural similarity of CARD-12 with other members of the Apaf-1/CED4family members suggests a possible mechanism for its proapoptoticactivity. In the case of both CARD-4 and Apaf-1, upstream signal-inducedself-oligomerization at the central NBS domain leads to the inducedproximity of effector molecules bound in heterotypic CARD/CARDinteractions. Apaf-1 interacts with procaspase-9 via a CARD/CARDinteraction. Oligomerization of Apaf-1 in response to mitochondrialcytochrome-c release induces proximity of bound procaspase-9 molecules,which then autoactivate, leading to downstream activation of caspase-3.This “Induced Proximity Model” of protein activation has been proposedas a general mechanism of caspase activation and of signal transductionfor signaling partners such as CARD-4/RICK, Apaf-1/Caspase-9 andFADD/Caspase-8. Based on the structural and functional data presentedhere, it is reasonable to speculate that the proapoptotic activity ofCARD-12 may occur through oligomerization of CARD-12 molecules at thecentral NBS domain in response to an upstream stress signal.CARD-12-induced cell death may then proceed in a CARD-5-dependent orpossibly caspase-1 dependent path. Similarly, CARD-12 may be involved inproinflammatory signaling by influencing caspase-1 activation ofinterleukin-1beta. TABLE 1 Summary of Human CARD-12 Sequence InformationSource of CARD-12 Predicted Predicted Predicted Sequence cDNA ProteinORF FIG. cDNA sequence SEQ ID SEQ ID SEQ ID NO:1 NO:2 NO:3 1E genomicsequence SEQ ID SEQ ID SEQ ID NO:4 NO:5 NO:6 2E

[0095] TABLE 2 Summary of Domains of CARD-12 Domain Location in CARD-12CARD about amino acids 1-88 of SEQ ID NO:2 NBS about amino acids 169-456of SEQ ID NO:2 NACHT about amino acids 169-186 (P-Loop/Walker A Box/Motif I); 196-220 (Walker B Box/Motif II); 229-253 (Motif III); 261-282(Motif IV); 330-351 (Motif V); 414-430 (Motif VI) and 438-457 (MotifVII) of SEQ ID NO:2 Neuronal AIP about amino acids 150-1024 of SEQ IDNO:2 Homology Leucine rich about amino acids 656-686, 687-708, 711-737,738- repeats 761, 762-788, 789-817, 819-845, 846-874, 875-901, 903-930,936-962, 965-993, and 994-1021 of SEQ ID NO:2. LRR Domain about aminoacids 656-1021 of SEQ ID NO:2

[0096] A plasmid containing a ______ encoding human CARD-12 (p ______)was deposited with the American Type Culture Collection (ATCC),Manasass, Va. on ______, 1999, and assigned Accession Number ______.This deposit will be maintained under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure. This deposit was made merely as aconvenience for those of skill in the art and is not an admission that adeposit is required under 35 U.S.C. §112.

[0097] A region, the CARD domain, of human CARD-12 protein (SEQ ID NO:2)bears some similarity to the CARD domains of CARD-3, CARD-4, CARD-5,CARD-6, CARD-7, CARD-8, CARD-9, CARD-10, CARD-11, CARD-13, CARD-14, andCARD-15. Detailed information concerning CARD-3, CARD-4, CARD-5, CARD-6,CARD-7, CARD-8, CARD-9, CARD-10, CARD-11, CARD-13, CARD-14, and CARD-15,can be found in U.S. application Ser. No. 09/245,281, filed Feb. 5,1999, U.S. application Ser. No. 09/207,359, filed Dec. 8, 1998, U.S.application Ser. No. 09/099,041, filed Jun. 17, 1998, U.S. applicationSer. No. 09/019,942, filed Feb. 6, 1998, U.S. application Ser. No.09/428,252, filed Oct. 27, 1999, U.S. application Ser. No. 60/180,021,filed Feb. 3, 2000, U.S. application Ser. No. 09/573,641, filed May 17,2000, U.S. application Ser. No. 60/181,159 filed Feb. 9, 2000, U.S.application Ser. No. 60/168,780 filed Dec. 3, 1999, U.S. applicationSer. No. 09/507,533 filed Feb. 18, 2000, and U.S. application Ser. No.09/513,904 filed Feb. 25, 2000. The entire content of each of theseapplications is incorporated herein by reference.

[0098] Human CARD-12 is a member of a family of molecules (the CARD-12family) having certain conserved structural and functional features. Theterm “family” when referring to the protein and nucleic acid moleculesof the invention is intended to mean two or more proteins or nucleicacid molecules having a common structural domain and having sufficientamino acid or nucleotide sequence identity as defined herein. Suchfamily members can be naturally occurring and can be from either thesame or different species. For example, a family can contain a firstprotein of human origin and a homologue of that protein of murineorigin, as well as a second, distinct protein of human origin and amurine homologue of that protein. Members of a family may also havecommon functional characteristics.

[0099] Preferred CARD-12 polypeptides of the present invention includean amino acid sequence sufficiently identical to one or more of thefollowing domains: a CARD domain, an NBS domain, and a LRR domain.

[0100] As used herein, the term “sufficiently identical” refers to afirst amino acid or nucleotide sequence which contains a sufficient orminimum number of identical or equivalent (e.g., an amino acid residuewhich has a similar side chain) amino acid residues or nucleotides to asecond amino acid or nucleotide sequence such that the first and secondamino acid or nucleotide sequences have a common structural domainand/or common functional activity. For example, amino acid or nucleotidesequences which contain a common structural domain having about 65%identity, preferably 75% identity, more preferably 85%, 95%, or 98%identity are defined herein as sufficiently identical.

[0101] As used interchangeably herein a “CARD-12 activity”, “biologicalactivity of CARD-12” or “functional activity of CARD-12”, refers to anactivity exerted by a CARD-12 protein, polypeptide or nucleic acidmolecule on a CARD-12 responsive cell as determined in vivo, or invitro, according to standard techniques. CARD-12 may act as apro-apoptotic protein or an anti-apoptotic protein (i.e., it might actto decrease or increase apoptosis). A CARD-12 activity can be a directactivity, such as an association with or an enzymatic activity on asecond protein or an indirect activity, such as a cellular signalingactivity mediated by interaction of the CARD-12 protein with a secondprotein.

[0102] In one embodiment, a CARD-12 activity can include at least one ormore of the following activities: (i) the ability to interact withproteins in an apoptotic signaling pathway (ii) the ability to interactwith a CARD-12 ligand; or (iii) the ability to interact with anintracellular target protein; (iv) the ability to interact, directly orindirectly with one or more with proteins having a CARD domain, e.g., acaspase or an AIP (e.g., AIP-I or AIP-2); (v) the ability to modulatethe activity of a caspase, e.g., caspase-9; (vi) the ability to modulatethe activity of NF-κB; (vii) the ability to modulate Apaf-1; (viii) theability to interact directly or indirectly with a Bcl-2 family member;(ix) the ability to modulate the activity of a stress activated kinase(e.g., JNK/p38); and (x) the ability to modulate phosphorylation of CHOP(GADD 153). CARD-12 nucleic acid and polypeptides as well as modulatorsof activity of expression of CARD-12 might be used to modulate an Apaf-1signaling pathway. CARD-12 may modulate the activity of a neurotrophinreceptor and thus modulate apoptosis of neuronal cells. Accordingly,CARD-12 nucleic acids and polypeptides as well as modulators of CARD-12activity or expression can be used to modulate apoptosis of neurons(e.g., for treatment of neurological disorders, particularlyneurodegenerative disorders).

[0103] Accordingly, another embodiment of the invention featuresisolated CARD-12 proteins and polypeptides having a CARD-12 activity.

[0104] Various aspects of the invention are described in further detailin the following subsections.

[0105] I. Isolated Nucleic Acid Molecules

[0106] One aspect of the invention pertains to isolated nucleic acidmolecules that encode CARD-12 proteins or biologically active portionsthereof, as well as nucleic acid molecules sufficient for use ashybridization probes to identify CARD-12-encoding nucleic acids (e.g.,CARD-12 mRNA) and fragments for use as PCR primers for the amplificationor mutation of CARD-12 nucleic acid molecules. As used herein, the term“nucleic acid molecule” is intended to include DNA molecules (e.g., cDNAor genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

[0107] An “isolated” nucleic acid molecule is one which is separatedfrom other nucleic acid molecules which are present in the naturalsource of the nucleic acid. Preferably, an “isolated” nucleic acid isfree of sequences (preferably protein encoding sequences) that whichnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated CARD-12 nucleic acid molecule can contain less than about 5kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequenceswhich naturally flank the nucleic acid molecule in genomic DNA of thecell from which the nucleic acid is derived. Moreover, an “isolated”nucleic acid molecule, such as a cDNA molecule, can be substantiallyfree of other cellular material, or culture medium when produced byrecombinant techniques, or substantially free of chemical precursors orother chemicals when chemically synthesized.

[0108] A nucleic acid molecule of the present invention, e.g., a nucleicacid molecule having the nucleotide sequence of SEQ ID NO:1, SEQ IDNO:3, the cDNA of ATCC ______, or a complement of any of thesenucleotide sequences, can be isolated using standard molecular biologytechniques and the sequence information provided herein. Using all orportion of the nucleic acid sequences of SEQ ID NO:1, SEQ ID NO:3, thecDNA of ATCC ______, as a hybridization probe, CARD-12 nucleic acidmolecules can be isolated using standard hybridization and cloningtechniques (e.g., as described in Sambrook et al., eds., MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0109] A nucleic acid of the invention can be amplified using cDNA, mRNAor genomic DNA as a template and appropriate oligonucleotide primersaccording to standard PCR amplification techniques. The nucleic acid soamplified can be cloned into an appropriate vector and characterized byDNA sequence analysis. Furthermore, oligonucleotides corresponding toCARD-12 nucleotide sequences can be prepared by standard synthetictechniques, e.g., using an automated DNA synthesizer.

[0110] In another embodiment, an isolated nucleic acid molecule of theinvention comprises a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, the cDNA of ATCC, or a portion thereof. A nucleic acid molecule which is complementaryto a given nucleotide sequence is one which is sufficientlycomplementary to the given nucleotide sequence that it can hybridize tothe given nucleotide sequence thereby forming a stable duplex.

[0111] Moreover, the nucleic acid molecule of the invention can compriseonly a portion of a nucleic acid sequence encoding CARD-12, for example,a fragment which can be used as a probe or primer or a fragment encodinga biologically active portion of CARD-12. The nucleotide sequencedetermined from the cloning of the human CARD-12 gene allows for thegeneration of probes and primers designed for use in identifying and/orcloning CARD-12 homologues in other cell types, e.g., from othertissues, as well as CARD-12 homologues and orthologs from other mammals.The probe/primer typically comprises substantially purifiedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, more preferably about 50, 75, 100,125, 150, 175, 200, 250, 300, 350 or 400 consecutive nucleotides of thesense or anti-sense sequence of SEQ ID NO:1, SEQ ID NO:3, the cDNA ofATCC ______, or of a naturally occurring mutant of one of SEQ ID NO:1,SEQ ID NO:3, or the cDNA of ATCC ______.

[0112] Probes based on the CARD-12 nucleotide sequence can be used todetect transcripts or genomic sequences encoding the same or similarproteins. The probe comprises a label group attached thereto, e.g., aradioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.Such probes can be used as a part of a diagnostic test kit foridentifying allelic variants and orthologs of the CARD-12 proteins ofthe present invention, identifying cells or tissue which mis-express aCARD-12 protein, such as by measuring a level of a CARD-12-encodingnucleic acid in a sample of cells from a subject, e.g., detectingCARD-12 mRNA levels or determining whether a genomic CARD-12 gene hasbeen mutated or deleted.

[0113] A nucleic acid fragment encoding a “biologically active portion”of CARD-12 can be prepared by isolating a portion of SEQ ID NO:1, SEQ IDNO:3, or the cDNA of ATCC ______, which encodes a polypeptide having aCARD-12 biological activity, expressing the encoded portion of CARD-12protein (e.g., by recombinant expression in vitro) and assessing theactivity of the encoded portion of CARD-12. For example, a nucleic acidfragment encoding a biologically active portion of CARD-12 includes aCARD domain, e.g., amino acids 1-88 of SEQ ID NO:2.

[0114] The invention further encompasses nucleic acid molecules thatdiffer from the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, and thecDNA of ATCC ______, due to degeneracy of the genetic code and thusencode the same CARD-12 protein as that encoded by the nucleotidesequence shown in SEQ ID NO:1, SEQ ID NO:3, or the cDNA of ATCC ______.

[0115] In addition to the CARD-12 nucleotide sequence shown in SEQ IDNO:1, SEQ ID NO:3, and the cDNA of ATCC ______, it will be appreciatedby those skilled in the art that DNA sequence polymorphisms that lead tochanges in the amino acid sequences of CARD-12 may exist within apopulation (e.g., the human population). Such genetic polymorphism inthe CARD-12 gene may exist among individuals within a population due tonatural allelic variation. As used herein, the terms “gene” and“recombinant gene” refer to nucleic acid molecules comprising an openreading frame encoding a CARD-12 protein, preferably a mammalian CARD-12protein. Such natural allelic variations can typically result in 1-5%variance in the nucleotide sequence of the CARD-12 gene. Any and allsuch nucleotide variations and resulting amino acid polymorphisms inCARD-12 that are the result of natural allelic variation and that do notalter the functional activity of CARD-12 are intended to be within thescope of the invention. Thus, e.g., 1%, 2%, 3%, 4%, or 5% of the aminoacids in CARD-12 (e.g., 1, 2, 3, 4, 5, 6, 8, 10, 15, 20, or fewer aminoacids) are replaced by another amino acid, preferably by conservativesubstitution.

[0116] Moreover, nucleic acid molecules encoding CARD-12 proteins fromother species (CARD-12 orthologs/homologues), which have a nucleotidesequence which differs from that of a CARD-12 disclosed herein, areintended to be within the scope of the invention.

[0117] Accordingly, in another embodiment, an isolated nucleic acidmolecule of the invention is at least 150 (300, 325, 350, 375, 400, 425,450, 500, 550, 600, 650, 700, 800, 900, 1000, 1300, 1600, 1900, 2100,2400, 2700, 3000, or 3100) nucleotides in length and hybridizes understringent conditions to the nucleic acid molecule comprising thenucleotide sequence, preferably the coding sequence, of SEQ ID NO:1, SEQID NO:3, or the cDNA of ATCC ______.

[0118] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% (65%, 70%, preferably 75%)identical to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6. An, non-limiting example of stringent hybridizationconditions are hybridization in 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at50-65° C. (e.g., 50° C. or 60° C. or 65° C.). Preferably, the isolatednucleic acid molecule of the invention that hybridizes under stringentconditions corresponds to a naturally-occurring nucleic acid molecule.As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs in ahuman cell in nature (e.g., encodes a natural protein).

[0119] In addition to naturally-occurring allelic variants of theCARD-12 sequence that may exist in the population, the skilled artisanwill further appreciate that changes can be introduced by mutation intothe nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or the cDNA of ATCC______, thereby leading to changes in the amino acid sequence of theencoded protein without altering the functional ability of the protein.For example, one can make nucleotide substitutions leading to amino acidsubstitutions at “non-essential” amino acid residues. A “non-essential”amino acid residue is a residue that can be altered from the wild-typesequence of CARD-12 protein without altering the biological activity,whereas an “essential” amino acid residue is required for biologicalactivity. For example, amino acid residues that are conserved among theCARD-12, proteins of various species are predicted to be particularlyunamenable to alteration.

[0120] For example, preferred CARD-12 proteins of the present inventioncontain at least one CARD domain. Additionally, a CARD-12 protein alsocontains at least one kinase-2 domain, at least one P-loop domain, atleast one nucleotide binding site domain, and at least one LRR domain.Such conserved domains are less likely to be amenable to mutation. Otheramino acid residues, however, (e.g., those that are not conserved oronly semi-conserved among CARD-12 of various species) may not beessential for activity and thus are likely to be amenable to alteration.

[0121] Accordingly, another aspect of the invention pertains to nucleicacid molecules encoding CARD-12 proteins that contain changes in aminoacid residues that are not essential for activity. Such CARD-12 proteinsdiffer in amino acid sequence from SEQ ID NO:2, and yet retainbiological activity. In one embodiment, the isolated nucleic acidmolecule includes a nucleotide sequence encoding a protein that includesan amino acid sequence that is at least about 45% identical, 65%, 75%,85%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO:2. Anisolated nucleic acid molecule encoding a CARD-12 protein having asequence which differs from that of SEQ ID NO:1, SEQ ID NO:3, or cDNA ofATCC ______, can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequence ofCARD-12 (SEQ ID NO:1, SEQ ID NO:3, the cDNA of ATCC ______) such thatone or more amino acid substitutions, additions or deletions areintroduced into the encoded protein. Mutations can be introduced bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. Thus, forexample, 1%, 2%, 3%, 5%, or 10% of the amino acids can be replaced byconservative substitution. A “conservative amino acid substitution” isone in which the amino acid residue is replaced with an amino acidresidue having a similar side chain. Families of amino acid residueshaving similar side chains have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in CARD-12 is preferably replaced with another aminoacid residue from the same side chain family. Alternatively, mutationscan be introduced randomly along all or part of a CARD-12 codingsequence, such as by saturation mutagenesis, and the resultant mutantscan be screened for CARD-12 biological activity to identify mutants thatretain activity. Following mutagenesis, the encoded protein can beexpressed recombinantly and the activity of the protein can bedetermined.

[0122] In an embodiment, a mutant CARD-12 protein can be assayed for:(1) the ability to form protein:protein interactions with proteins inthe apoptotic signaling pathway; (2) the ability to bind a CARD-12ligand; or (3) the ability to bind to an intracellular target protein.

[0123] The present invention encompasses antisense nucleic acidmolecules, i.e., molecules which are complementary to a sense nucleicacid encoding a protein, e.g., complementary to the coding strand of adouble-stranded cDNA molecule or complementary to an mRNA sequence.Accordingly, an antisense nucleic acid can hydrogen bond to a sensenucleic acid. The antisense nucleic acid can be complementary to anentire CARD-12 coding strand, or to only a portion thereof, e.g., all orpart of the protein coding region (or open reading frame). An antisensenucleic acid molecule can be antisense to a noncoding region of thecoding strand of a nucleotide sequence encoding CARD-12. The noncodingregions (“5′ and 3′ untranslated regions”) are the 5′ and 3′ sequencesthat flank the coding region and are not translated into amino acids.

[0124] Given the coding strand sequences encoding CARD-12 disclosedherein, antisense nucleic acids of the invention can be designedaccording to the rules of Watson and Crick base pairing. The antisensenucleic acid molecule can be complementary to the entire coding regionof CARD-12 mRNA, but more preferably is an oligonucleotide which isantisense to only a portion of the coding or noncoding region of CARD-12mRNA. For example, the antisense oligonucleotide can be complementary tothe region surrounding the translation start site of CARD-12 mRNA. Anantisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25,30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid ofthe invention can be constructed using chemical synthesis and enzymaticligation reactions using procedures known in the art. For example, anantisense nucleic acid (e.g., an antisense oligonucleotide) can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense nucleic acid include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-aino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

[0125] The antisense nucleic acid molecules of the invention aretypically administered to a subject or generated in situ such that theyhybridize with or bind to cellular mRNA and/or genomic DNA encoding aCARD-12 protein to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An antisense nucleic acid molecule of the inventioncan be administered by direct injection at a tissue site. Alternatively,antisense nucleic acid molecules can be modified to target selectedcells and then administered systemically. For example, for systemicadministration, antisense molecules can be modified such that theyspecifically bind to receptors or antigens expressed on a selected cellsurface, e.g., by linking the antisense nucleic acid molecules topeptides or antibodies which bind to cell surface receptors or antigens.The antisense nucleic acid molecules can also be delivered to cellsusing the vectors described herein. To achieve sufficient intracellularconcentrations of the antisense molecules, vector constructs in whichthe antisense nucleic acid molecule is placed under the control of astrong pol II or pol III promoter are preferred.

[0126] An antisense nucleic acid molecule of the invention can be anα-anomeric nucleic acid molecule. An α-anomeric nucleic acid moleculeforms specific double-stranded hybrids with complementary RNA in which,contrary to the usual β-units, the strands run parallel to each other(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisensenucleic acid molecule can also comprise a 2′-o-methylribonucleotide(Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimericRNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

[0127] The invention also encompasses ribozymes. Ribozymes are catalyticRNA molecules with ribonuclease activity which are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region. Thus, ribozymes (e.g., hammerhead ribozymes(described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can beused to catalytically cleave CARD-12 mRNA transcripts to thereby inhibittranslation of CARD-12 mRNA. A ribozyme having specificity for aCARD-12-encoding nucleic acid can be designed based upon the nucleotidesequence of a CARD-12 cDNA disclosed herein. For example, a derivativeof a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in a CARD-12-encoding mRNA. See, e.g., Cech et al. U.S.Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742.Alternatively, CARD-12 mRNA can be used to select a catalytic RNA havinga specific ribonuclease activity from a pool of RNA molecules. See,e.g., Bartel and Szostak (1993) Science 261:1411-1418.

[0128] The invention also encompasses nucleic acid molecules which formtriple helical structures. For example, CARD-12 gene expression can beinhibited by targeting nucleotide sequences complementary to theregulatory region of the CARD-12 (e.g., the CARD-12 promoter and/orenhancers) to form triple helical structures that prevent transcriptionof the CARD-12 gene in target cells. See generally, Helene (1991)Anticancer Drug Des. 6(6):569-84; Helene (1992) Ann. N.Y. Acad. Sci.660:27-36; and Maher (1992) Bioassays 14(12):807-15.

[0129] In embodiments, the nucleic acid molecules of the invention canbe modified at the base moiety, sugar moiety or phosphate backbone toimprove, e.g., the stability, hybridization, or solubility of themolecule. For example, the deoxyribose phosphate backbone of the nucleicacids can be modified to generate peptide nucleic acids (see Hyrup etal. (1996) Bioorganic & Medicinal Chemistry 4(1):5-23). As used herein,the terms “peptide nucleic acids” or “PNAs” refer to nucleic acidmimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone isreplaced by a pseudopeptide backbone and only the four naturalnucleobases are retained. The neutral backbone of PNAs has been shown toallow for specific hybridization to DNA and RNA under conditions of lowionic strength. The synthesis of PNA oligomers can be performed usingstandard solid phase peptide synthesis protocols as described in Hyrupet al. (1996) supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci.USA 93:14670-675.

[0130] PNAs of CARD-12 can be used for therapeutic and diagnosticapplications. For example, PNAs can be used as antisense or antigeneagents for sequence-specific modulation of gene expression by, e.g.,inducing transcription or translation arrest or inhibiting replication.PNAs of CARD-12 can also be used, e.g., in the analysis of single basepair mutations in a gene by, e.g., PNA directed PCR clamping; asartificial restriction enzymes when used in combination with otherenzymes, e.g., S1 nucleases (Hyrup (1996) supra; or as probes or primersfor DNA sequence and hybridization (Hyrup (1996) supra; Perry-O'Keefe etal. (1996) Proc. Natl. Acad. Sci. USA 93: 14670-675).

[0131] In another embodiment, PNAs of CARD-12 can be modified, e.g., toenhance their stability or cellular uptake, by attaching lipophilic orother helper groups to PNA, by the formation of PNA-DNA chimeras, or bythe use of liposomes or other techniques of drug delivery known in theart. For example, PNA-DNA chimeras of CARD-12 can be generated which maycombine the advantageous properties of PNA and DNA. Such chimeras allowDNA recognition enzymes, e.g., RNAse H and DNA polymerases, to interactwith the DNA portion while the PNA portion would provide high bindingaffinity and specificity. PNA-DNA chimeras can be linked using linkersof appropriate lengths selected in terms of base stacking, number ofbonds between the nucleobases, and orientation (Hyrup (1996) supra). Thesynthesis of PNA-DNA chimeras can be performed as described in Hyrup(1996) supra and Finn et al. (1996) Nucleic Acids Research24(17):3357-63. For example, a DNA chain can be synthesized on a solidsupport using standard phosphoramidite coupling chemistry and modifiednucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite, can be used as a between the PNA and the 5′ end of DNA(Mag et al. (1989) Nucleic Acid Res. 17:5973-88). PNA monomers are thencoupled in a stepwise manner to produce a chimeric molecule with a 5′PNA segment and a 3′ DNA segment (Finn et al. (1996) Nucleic AcidsResearch 24(17):3357-63). Alternatively, chimeric molecules can besynthesized with a 5′ DNA segment and a 3′ PNA segment (Peterser et al.(1975) Bioorganic Med. Chem. Lett. 5:1119-11124).

[0132] In other embodiments, the oligonucleotide may include otherappended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA84:648-652; PCT Publication No. WO 88/09810) or the blood-brain barrier(see, e.g., PCT Publication No. WO 89/10134). In addition,oligonucleotides can be modified with hybridization-triggered cleavageagents (see, e.g., Krol et al. (1988) Bio/Techniques 6:958-976) orintercalating agents (see, e.g., Zon (1988) Pharm. Res. 5:539-549). Tothis end, the oligonucleotide may be conjugated to another molecule,e.g., a peptide, hybridization triggered cross-linking agent, transportagent, hybridization-triggered cleavage agent, etc.

[0133] II. Isolated CARD-12 Proteins and Anti-CARD-12 Antibodies.

[0134] One aspect of the invention pertains to isolated CARD-12proteins, and biologically active portions thereof, as well aspolypeptide fragments suitable for use as immunogens to raiseanti-CARD-12 antibodies. In one embodiment, native CARD-12 proteins canbe isolated from cells or tissue sources by an appropriate purificationscheme using standard protein purification techniques. In anotherembodiment, CARD-12 proteins are produced by recombinant DNA techniques.Alternative to recombinant expression, a CARD-12 protein or polypeptidecan be synthesized chemically using standard peptide synthesistechniques.

[0135] An “isolated” or “purified” protein or biologically activeportion thereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theCARD-12 protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations ofCARD-12 protein in which the protein is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. Thus, CARD-12 protein that is substantially free of cellularmaterial includes preparations of CARD-12 protein having less than about30%, 20%, 10%, or 5% (by dry weight) of non-CARD-12 protein (alsoreferred to herein as a “contaminating protein”). When the CARD-12protein or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, 10%, or 5% of thevolume of the protein preparation. When CARD-12 protein is produced bychemical synthesis, it is preferably substantially free of chemicalprecursors or other chemicals, i.e., it is separated from chemicalprecursors or other chemicals which are involved in the synthesis of theprotein. Accordingly such preparations of CARD-12 protein have less thanabout 30%, 20%, 10%, 5% (by dry weight) of chemical precursors ornon-CARD-12 chemicals.

[0136] Biologically active portions of a CARD-12 protein includepeptides comprising amino acid sequences sufficiently identical to orderived from the amino acid sequence of the CARD-12 protein (e.g., theamino acid sequence shown in SEQ ID NO:2), which include less aminoacids than the full length CARD-12 protein, and exhibit at least oneactivity of a CARD-12 protein. Typically, biologically active portionscomprise a domain or motif with at least one activity of the CARD-12protein. A biologically active portion of a CARD-12 protein can be apolypeptide which is, for example, 10, 25, 50, 100, 150, 200, 250, 300,400, 500, 600, 700, 800, 900, 1000 or more amino acids in length.Preferred biologically active polypeptides include one or moreidentified CARD-12 structural domains, e.g., the CARD domain (aminoacids 1-88 of SEQ ID NO:2).

[0137] Moreover, other biologically active portions, in which otherregions of the protein are deleted, can be prepared by recombinanttechniques and evaluated for one or more of the functional activities ofa native CARD-12 protein.

[0138] CARD-12 protein has the amino acid sequence shown of SEQ ID NO:2.Other useful CARD-12 proteins are substantially identical to SEQ ID NO:2and retain the functional activity of the protein of SEQ ID NO:2, yetdiffer in amino acid sequence due to natural allelic variation ormutagenesis.

[0139] A useful CARD-12 protein is a protein which includes an aminoacid sequence at least about 45%, preferably 55%, 65%, 75%, 85%, 95%, or99% identical to the amino acid sequence of SEQ ID NO:2, and retains thefunctional activity of the CARD-12 protein of SEQ ID NO:2.

[0140] To determine the percent identity of two amino acid sequences orof two nucleic acids, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoor nucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity =# ofidentical positions/total # of positions×100).

[0141] The determination of percent homology between two sequences canbe accomplished using a mathematical algorithm. A preferred,non-limiting example of a mathematical algorithm utilized for thecomparison of two sequences is the algorithm of Karlin and Altschul(1990) Proc. Nat'l Acad. Sci. USA 87:2264-2268, modified as in Karlinand Altschul (1993) Proc. Nat'l Acad. Sci. USA 90:5873-5877. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul, et al. (1990) J. Mol. Biol. 215:403-410. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences similar or homologous toCARD-12 nucleic acid molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example ofa mathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, CABIOS (1989). Such an algorithm isincorporated into the ALIGN program (version 2.0) which is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.When utilizing the ALIGN program for comparing nucleic acid sequences, agap length penalty of 12, and a gap penalty of 4 can be used.

[0142] The percent identity between two sequences can be determinedusing techniques similar to those described above, with or withoutallowing gaps. In calculating percent identity, typically exact matchesare counted.

[0143] The invention also provides CARD-12 chimeric or fusion proteins.As used herein, a CARD-12 “chimeric protein” or “fusion protein”comprises a CARD-12 polypeptide operatively linked to a non-CARD-12polypeptide. A “CARD-12 polypeptide” refers to a polypeptide having anamino acid sequence corresponding to all or a portion (preferably abiologically active portion) of a CARD-12, whereas a “non-CARD-12polypeptide” refers to a polypeptide having an amino acid sequencecorresponding to a protein which is not substantially identical to theCARD-12 protein, e.g., a protein which is different from the CARD-12proteins and which is derived from the same or a different organism.Within the fusion protein, the term “operatively linked” is intended toindicate that the CARD-12 polypeptide and the non-CARD-12 polypeptideare fused in-frame to each other. The heterologous polypeptide can befused to the N-terminus or C-terminus of the CARD-12 polypeptide.

[0144] One useful fusion protein is a GST fusion protein in which theCARD-12 sequences are fused to the C-terminus of the GST sequences. Suchfusion proteins can facilitate the purification of recombinant CARD-12.In another embodiment, the fusion protein contains a signal sequencefrom another protein. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of CARD-12 can be increased throughuse of a heterologous signal sequence. For example, the gp67 secretorysequence of the baculovirus envelope protein can be used as aheterologous signal sequence (Current Protocols in Molecular Biology,Ausubel et al., eds., John Wiley & Sons, 1992). Other examples ofeukaryotic heterologous signal sequences include the secretory sequencesof melittin and human placental alkaline phosphatase (Stratagene; LaJolla, Calif.). In yet another example, useful prokaryotic heterologoussignal sequences include the phoA secretory signal (Molecular cloning,Sambrook et al, second edition, Cold spring harbor laboratory press,1989) and the protein A secretory signal (Pharmacia Biotech; Piscataway,N.J.).

[0145] In yet another embodiment, the fusion protein is aCARD-12-immunoglobulin fusion protein in which all or part of CARD-12 isfused to sequences derived from a member of the immunoglobulin proteinfamily. The CARD-12-immunoglobulin fusion proteins of the invention canbe incorporated into pharmaceutical compositions and administered to asubject to inhibit an interaction between a CARD-12 ligand and a CARD-12protein on the surface of a cell, to thereby suppress CARD-12-mediatedsignal transduction in vivo. The CARD-12-immunoglobulin fusion proteinscan be used to affect the bioavailability of a CARD-12 cognate ligand.Inhibition of the CARD-12 ligand/CARD-12 interaction may be usefultherapeutically for both the treatment of proliferative anddifferentiative disorders, as well as modulating (e.g., promoting orinhibiting) cell survival. Moreover, the CARD-12-immunoglobulin fusionproteins of the invention can be used as immunogens to produceanti-CARD-12 antibodies in a subject, to purify CARD-12 ligands and inscreening assays to identify molecules which inhibit the interaction ofCARD-12 with a CARD-12 ligand.

[0146] Preferably, a CARD-12 chimeric or fusion protein of the inventionis produced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, e.g., Current Protocols in MolecularBiology, Ausubel et al. eds., John Wiley & Sons: 1992). Moreover, manyexpression vectors are commercially available that already encode afusion moiety (e.g., a GST polypeptide). A CARD-12-encoding nucleic acidcan be cloned into such an expression vector such that the fusion moietyis linked in-frame to the CARD-12 protein.

[0147] The present invention also pertains to variants of the CARD-12proteins which function as either CARD-12 agonists (mimetics) or asCARD-12 antagonists. Variants of the CARD-12 protein can be generated bymutagenesis, e.g., discrete point mutation or truncation of the CARD-12protein. An agonist of the CARD-12 protein can retain substantially thesame, or a subset, of the biological activities of the naturallyoccurring form of the CARD-12 protein. An antagonist of the CARD-12protein can inhibit one or more of the activities of the naturallyoccurring form of the CARD-12 protein by, for example, competitivelybinding to a downstream or upstream member of a cellular signalingcCARD-5ade which includes the CARD-12 protein. Thus, specific biologicaleffects can be elicited by treatment with a variant of limited function.Treatment of a subject with a variant having a subset of the biologicalactivities of the naturally occurring form of the protein can have fewerside effects in a subject relative to treatment with the naturallyoccurring form of the CARD-12 proteins.

[0148] Variants of the CARD-12 protein which function as either CARD-12agonists (mimetics) or as CARD-12 antagonists can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutantsof the CARD-12 protein for CARD-12 protein agonist or antagonistactivity. In one embodiment, a variegated library of CARD-12 variants isgenerated by combinatorial mutagenesis at the nucleic acid level and isencoded by a variegated gene library. A variegated library of CARD-12variants can be produced by, for example, enzymatically ligating amixture of synthetic oligonucleotides into gene sequences such that adegenerate set of potential CARD-12 sequences is expressible asindividual polypeptides, or alternatively, as a set of larger fusionproteins (e.g., for phage display) containing the set of CARD-12sequences therein. There are a variety of methods which can be used toproduce libraries of potential CARD-12 variants from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be performed in an automatic DNA synthesizer, and thesynthetic gene then ligated into an appropriate expression vector. Useof a degenerate set of genes allows for the provision, in one mixture,of all of the sequences encoding the desired set of potential CARD-12sequences. Methods for synthesizing degenerate oligonucleotides areknown in the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura etal. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477).

[0149] Useful fragments of CARD-12, include fragments comprising orconsisting of a domain or subdomain described herein, e.g., a kinase-2domain or a CARD domain.

[0150] In addition, libraries of fragments of the CARD-12 protein codingsequence can be used to generate a variegated population of CARD-12fragments for screening and subsequent selection of variants of aCARD-12 protein. In one embodiment, a library of coding sequencefragments can be generated by treating a double stranded PCR fragment ofa CARD-12 coding sequence with a nuclease under conditions whereinnicking occurs only about once per molecule, denaturing the doublestranded DNA, renaturing the DNA to form double stranded DNA which caninclude sense/antisense pairs from different nicked products, removingsingle stranded portions from reformed duplexes by treatment with S1nuclease, and ligating the resulting fragment library into an expressionvector. By this method, an expression library can be derived whichencodes N-terminal and internal fragments of various sizes of theCARD-12 protein.

[0151] Several techniques are known in the art for screening geneproducts of combinatorial libraries made by point mutations ortruncation, and for screening cDNA libraries for gene products having aselected property. Such techniques are adaptable for rapid screening ofthe gene libraries generated by the combinatorial mutagenesis of CARD-12proteins. The most widely used techniques, which are amenable to highthrough-put analysis, for screening large gene libraries typicallyinclude cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates isolation of the vectorencoding the gene whose product was detected. Recursive ensemblemutagenesis (REM), a technique which enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify CARD-12 variants (Arkin and Yourvan (1992)Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) ProteinEngineering 6(3):327-331).

[0152] An isolated CARD-12 protein, or a portion or fragment thereof,can be used as an immunogen to generate antibodies that bind CARD-12using standard techniques for polyclonal and monoclonal antibodypreparation. The full-length CARD-12 protein can be used or,alternatively, the invention provides antigenic peptide fragments ofCARD-12 for use as immunogens. The antigenic peptide of CARD-12comprises at least 8 (preferably 10, 15, 20, or 30) amino acid residuesof the amino acid sequence shown in SEQ ID NO:2 and encompasses anepitope of CARD-12 such that an antibody raised against the peptideforms a specific immune complex with CARD-12.

[0153] Useful antibodies include antibodies which bind to a domain orsubdomain of CARD-12 described herein (e.g., a kinase-2 domain, a CARDdomain, an NBS domain, a P-loop domain, or a LRR domain).

[0154] Preferred epitopes encompassed by the antigenic peptide areregions of CARD-12 that are located on the surface of the protein, e.g.,hydrophilic regions. Other important criteria include a preference for aterminal sequence, high antigenic index (e.g., as predicted byJameson-Wolf algorithm), ease of peptide synthesis (e.g., avoidance ofprolines); and high surface probability (e.g., as predicted by the Eminialgorithm; FIG. 4).

[0155] A CARD-12 immunogen typically is used to prepare antibodies byimmunizing a suitable subject, (e.g., rabbit, goat, mouse or othermammal) with the immunogen. An appropriate immunogenic preparation cancontain, for example, recombinantly expressed CARD-12 protein or achemically synthesized CARD-12 polypeptide. The preparation can furtherinclude an adjuvant, such as Freund's complete or incomplete adjuvant,or similar immunostimulatory agent. Immunization of a suitable subjectwith an immunogenic CARD-12 preparation induces a polyclonalanti-CARD-12 antibody response.

[0156] Accordingly, another aspect of the invention pertains toanti-CARD-12 antibodies. The term “antibody” as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds an antigen, such as CARD-12. Amolecule which specifically binds to CARD-12 is a molecule which bindsCARD-12, but does not substantially bind other molecules in a sample,e.g., a biological sample, which naturally contains CARD-12. Examples ofimmunologically active portions of immunoglobulin molecules includeF(ab) and F(ab′)2 fragments which can be generated by treating theantibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies that bind CARD-12. The term“monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope of CARD-12. A monoclonal antibody composition thustypically displays a single binding affinity for a particular CARD-12protein with which it immunoreacts.

[0157] Polyclonal anti-CARD-12 antibodies can be prepared as describedabove by immunizing a suitable subject with a CARD-12 immunogen. Theanti-CARD-12 antibody titer in the immunized subject can be monitoredover time by standard techniques, such as with an enzyme linkedimmunosorbent assay (ELISA) using immobilized CARD-12. If desired, theantibody molecules directed against CARD-12 can be isolated from themammal (e.g., from the blood) and further purified by well-knowntechniques, such as protein A chromatography to obtain the IgG fraction.At an appropriate time after immunization, e.g., when the anti-CARD-12antibody titers are highest, antibody-producing cells can be obtainedfrom the subject and used to prepare monoclonal antibodies by standardtechniques, such as the hybridoma technique originally described byKohler and Milstein (1975) Nature 256:495-497, the human B cellhybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), theEBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. Thetechnology for producing various antibodies monoclonal antibodyhybridomas is well known (see generally Current Protocols in Immunology(1994) Coligan et al. (eds.) John Wiley & Sons, Inc., New York, N.Y.).Briefly, an immortal cell line (typically a myeloma) is fused tolymphocytes (typically splenocytes) from a mammal immunized with aCARD-12 immunogen as described above, and the culture supernatants ofthe resulting hybridoma cells are screened to identify a hybridomaproducing a monoclonal antibody that binds CARD-12.

[0158] Any of the many well known protocols used for fusing lymphocytesand immortalized cell lines can be applied for the purpose of generatingan anti-CARD-12 monoclonal antibody (see, e.g., Current Protocols inImmunology, supra; Galfre et al. (1977) Nature 266:55052; R. H. Kenneth,in Monoclonal Antibodies: A New Dimension In Biological Analyses, PlenumPublishing Corp., New York, N.Y. (1980); and Lerner (1981) Yale J. Biol.Med., 54:387-402). Moreover, the ordinarily skilled worker willappreciate that there are many variations of such methods which alsowould be useful. Typically, the immortal cell line (e.g., a myeloma cellline) is derived from the same mammalian species as the lymphocytes. Forexample, murine hybridomas can be made by fusing lymphocytes from amouse immunized with an immunogenic preparation of the present inventionwith an immortalized mouse cell line, e.g., a myeloma cell line that issensitive to culture medium containing hypoxanthine, aminopterin andthymidine (“HAT medium”). Any of a number of myeloma cell lines can beused as a fusion partner according to standard techniques, e.g., theP3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. Thesemyeloma lines are available from ATCC. Typically, HAT-sensitive mousemyeloma cells are fused to mouse splenocytes using polyethylene glycol(“PEG”). Hybridoma cells resulting from the fusion are then selectedusing HAT medium, which kills unfused and unproductively fused myelomacells (unfused splenocytes die after several days because they are nottransformed). Hybridoma cells producing a monoclonal antibody of theinvention are detected by screening the hybridoma culture supernatantsfor antibodies that bind CARD-12, e.g., using a standard ELISA assay.

[0159] Alternative to preparing monoclonal antibody-secretinghybridomas, a monoclonal anti-CARD-12 antibody can be identified andisolated by screening a recombinant combinatorial immunoglobulin library(e.g., an antibody phage display library) with CARD-12 to therebyisolate immunoglobulin library members that bind CARD-12. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, U.S. Pat. No. 5,223,409;PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCTPublication No. WO 92/20791; PCT Publication No. WO 92/15679; PCTPublication No. WO 93/01288; PCT Publication No. WO 92/01047; PCTPublication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs etal. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod.Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffithset al. (1993) EMBO J. 12:725-734.

[0160] Additionally, recombinant anti-CARD-12 antibodies, such aschimeric and humanized monoclonal antibodies, comprising both human andnon-human portions, which can be made using standard recombinant DNAtechniques, are within the scope of the invention. Such chimeric andhumanized monoclonal antibodies can be produced by recombinant DNAtechniques known in the art, for example using methods described in PCTPublication No. WO 87/02671; European Patent Application 184,187;European Patent Application 171,496; European Patent Application173,494; PCT Publication No. WO 86/01533; U.S. Pat. No. 4,816,567;European Patent Application 125,023; Better et al. (1988) Science240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al.(1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987)Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shawet al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, (1985)Science 229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; U.S. Pat.No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al.(1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.141:4053-4060.

[0161] An anti-CARD-12 antibody (e.g., monoclonal antibody) can be usedto isolate CARD-12 by standard techniques, such as affinitychromatography or immunoprecipitation. An anti-CARD-12 antibody canfacilitate the purification of natural CARD-12 from cells and ofrecombinantly produced CARD-12 expressed in host cells. Moreover, ananti-CARD-12 antibody can be used to detect CARD-12 protein (e.g., in acellular lysate or cell supernatant) in order to evaluate the abundanceand pattern of expression of the CARD-12 protein. Anti-CARD-12antibodies can be used diagnostically to monitor protein levels intissue as part of a clinical testing procedure, e.g., to, for example,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

[0162] Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

[0163] The conjugates of the invention can be used for modifying a givenbiological response. The drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, a-interferon, β-interferon, nerve growth factor,platelet derived growth factor, tissue plasminogen activator; or,biological response modifiers such as, for example, lymphokines,interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6),granulocyte macrophase colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF), or other growth factors.

[0164] Techniques for conjugating such therapeutic moiety to antibodiesare well known, see, e.g., Arnon et al., “Monoclonal Antibodies forImmunotargeting of Drugs in Cancer Therapy”, in Monoclonal Antibodiesand Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies for Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers of CytotoxicAgents in Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological and Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, and Future Prospective of TheTherapeutic Use of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation and Cytotoxic Properties of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980.

[0165] In addition, antibodies of the invention, either conjugated ornot conjugated to a therapeutic moiety, can be administered together orin combination with a therapeutic moiety such as a cytotoxin, atherapeutic agent or a radioactive metal ion. The order ofadministration of the antibody and therapeutic moiety can vary. Forexample, in some embodiments, the antibody is administered concurrently(through the same or different delivery devices, e.g., syringes) withthe therapeutic moiety. Alternatively, the antibody can be administeredseparately and prior to the therapeutic moiety. Still alternatively, thetherapeutic moiety is administered separately and prior to the antibody.In many embodiments, these administration regimens will be continued fordays, months or years.

[0166] Another aspect of the invention relates to a method for inducingan immunological response in a mammal which comprises inoculating themammal with a CARD-12 polypeptide, adequate to produce antibody and/or Tcell immune response to protect the animal from the diseaseshereinbefore mentioned, amongst others. Yet another aspect of theinvention relates to a method of inducing immunological response in amammal which comprises, delivering a CARD-12 polypeptide via a vectordirecting expression of the polynucleotide and coding for thepolypeptide in vivo in order to induce such an immunological response toproduce antibody to protect the animal from diseases.

[0167] A further aspect of the invention relates to animmunological/vaccine formulation (composition) which, when introducedinto a mammalian host, induces an immunological response in that mammalto a CARD-12 polypeptide of the present invention wherein thecomposition comprises a polypeptide or polynucleotide of CARD-12. Thevaccine formulation may further comprise a suitable carrier. Since apolypeptide may be broken down in the stomach, it is preferablyadministered parenterally (for instance, subcutaneous, intramuscular,intravenous, or intradermal injection). Formulations suitable forparenteral administration include aqueous and non-aqueous sterileinjection solutions which may contain anti-oxidants, buffers,bacteriostats and solutes which render the formulation instonic with theblood of the recipient; and aqueous and non-aqueous sterile suspensionswhich may include suspending agents or thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample, sealed ampoules and vials and may be stored in a freeze-driedcondition requiring only the addition of the sterile liquid carrierimmediately prior to use. The vaccine formulation may also includeadjuvant systems for enhancing the immunogenicity of the formulation,such as oil-in water systems and other systems known in the art. Thedosage will depend on the specific activity of the vaccine and can bereadily determined by routine experimentation.

[0168] III. Computer Readable Means

[0169] The nucleotide or amino acid sequences of the invention are alsoprovided in a variety of mediums to facilitate use thereof. As usedherein, “provided” refers to a manufacture, other than an isolatednucleic acid or amino acid molecule, which contains a nucleotide oramino acid sequence of the present invention. Such a manufactureprovides the nucleotide or amino acid sequences, or a subset thereof(e.g., a subset of open reading frames (ORFs)) in a form which allows askilled artisan to examine the manufacture using means not directlyapplicable to examining the nucleotide or amino acid sequences, or asubset thereof, as they exist in nature or in purified form.

[0170] In one application of this embodiment, a nucleotide or amino acidsequence of the present invention can be recorded on computer readablemedia. As used herein, “computer readable media” refers to any mediumthat can be read and accessed directly by a computer. Such mediainclude, but are not limited to: magnetic storage media, such as floppydiscs, hard disc storage medium, and magnetic tape; optical storagemedia such as CD-ROM; electrical storage media such as RAM and ROM; andhybrids of these categories such as magnetic/optical storage media. Thisskilled artisan will readily appreciate how any of the presently knowncomputer readable mediums can be used to create a manufacture comprisingcomputer readable medium having recorded thereon a nucleotide or aminoacid sequence of the present invention.

[0171] As used herein, “recorded” refers to a process for storinginformation on computer readable medium. The skilled artisan can readilyadopt any of the presently known methods for recording information oncomputer readable medium to generate manufactures comprising thenucleotide or amino acid sequence information of the present invention.

[0172] A variety of data storage structures are available to a skilledartisan for creating a computer readable medium having recorded thereona nucleotide or amino acid sequence of the present invention. The choiceof the data storage structure will generally be based on the meanschosen to access the stored information. In addition, a variety of dataprocessor programs and formats can be used to store the nucleotidesequence information of the present invention on computer readablemedium. The sequence information can be represented in a work processingtest file, formatted in commercially-available software such asWordPerfect and Microsoft Word, or represented in the form of anCARD-5II file, stored in a database application, such as DB2, Sybase,Oracle, or the like. The skilled artisan can readily adapt any number ofdata processor structuring formats (e.g., text file or database) inorder to obtain computer readable medium having recorded thereon thenucleotide sequence information of the present invention.

[0173] By providing the nucleotide or amino acid sequences of theinvention in computer readable form, the skilled artisan can routinelyaccess the sequence information for a variety of purposes. For example,one skilled in the art can use the nucleotide or amino acid sequences ofthe invention in computer readable form to compare a target sequence ora target structural motif with the sequence information stored withinthe data storage means. Search means are used to identify fragments orregions of the sequences of the invention which match a particulartarget sequence or target motif.

[0174] As used herein, a “target sequence” can be any DNA or amino acidsequence of six or more nucleotides or two or more amino acids. Askilled artisan can readily recognize that the longer a target sequenceis, the less likely a target sequence will be present as a randomoccurrence in the database. The most preferred sequence length of atarget sequence is from about 10 to 100 amino acids or from about 30 to300 nucleotide residues. However, it is well recognized thatcommercially important fragments, such as sequence fragments involved ingene expression and protein processing, may be of shorter length.

[0175] As used herein, “a target structural motif,” or “target motif,”refers to any rationally selected sequence or combination of sequencesin which the sequence(s) are chosen based on a three-dimensionalconfiguration formed upon the folding of the target motif. There are avariety of target motifs know in the art. Protein target motifs include,but are not limited to, enzyme active sites and signal sequences.Nucleic acid target motifs include, but are not limited to, promotersequences, hairpin structures and inducible expression elements (proteinbinding sequences).

[0176] Computer software is publicly available which allows a skilledartisan to access sequence information provided in a computer readablemedium for analysis and comparison to other sequences. A variety of knowalgorithms are disclosed publicly and a variety of commerciallyavailable software for conducting search means are and can be used inthe computer-based systems of the present invention. Examples of suchsoftware include, but is not limited to, MacPattern (EMBL), BLASTIN andBLASTX (NCBIA).

[0177] For example, software that implements the BLAST (Altschul et al.(1990) J. of Mol. Biol. 215:403-410) and BLAZE (Brutlag et al. (1993)Comp. Chem. 17:203-207) search algorithms on a Sybase system can be usedto identify open reading frames (ORFs) of the sequences of the inventionwhich contain homology to ORFs or proteins from other libraries. SuchORFs are protein-encoding fragments and are useful in producingcommercially important proteins such as enzymes used in variousreactions and in the production of commercially useful metabolites.

[0178] IV. Recombinant Expression Vectors and Host Cells

[0179] Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding CARD-12 (or aportion thereof). As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors, expressionvectors, are capable of directing the expression of genes to which theyare operatively linked. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids (vectors).However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

[0180] The recombinant expression vectors of the invention comprise anucleic acid of the invention in a form suitable for expression of thenucleic acid in a host cell, which means that the recombinant expressionvectors include one or more regulatory sequences, selected on the basisof the host cells to be used for expression, which is operatively linkedto the nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory sequence(s)in a manner which allows for expression of the nucleotide sequence(e.g., in an in vitro transcription/translation system or in a host cellwhen the vector is introduced into the host cell). The term “regulatorysequence” is intended to include promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcell and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences). Itwill be appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, etc.The expression vectors of the invention can be introduced into hostcells to thereby produce proteins or peptides, including fusion proteinsor peptides, encoded by nucleic acids as described herein (e.g., CARD-12proteins, mutant forms of CARD-12, fusion proteins, etc.).

[0181] The recombinant expression vectors of the invention can bedesigned for expression of CARD-12 in prokaryotic or eukaryotic cells,e.g., bacterial cells such as E. coli, insect cells (using baculovirusexpression vectors) yeast cells or mammalian cells. Suitable host cellsare discussed further in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

[0182] Expression of proteins in prokaryotes is most often carried outin E. coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith and Johnson (1988) Gene 67:31-40), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein.

[0183] Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., Gene Expression Technology: Methods in Enzymology 185,Academic Press, San Diego, Calif. (1990) 60-89). Target gene expressionfrom the pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gnl 0-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gnl). This viralpolymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from aresident ë prophage harboring a T7 gnl gene under the transcriptionalcontrol of the lacUV5 promoter.

[0184] One strategy to maximize recombinant protein expression in E.coli is to express the protein in a bacterial having an impairedcapacity to proteolytically cleave the recombinant protein (Gottesman,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, California (1990) 119-128). Another strategy is to alter thenucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in E. coli (Wada et al. (1992) NucleicAcids Res. 20:2111-2118). Such alteration of nucleic acid sequences ofthe invention can be carried out by standard DNA synthesis techniques.

[0185] In another embodiment, the CARD-12 expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerivisae include pYepSec1 Baldari et al. (1987) EMBO J. 6:229-234),pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz etal. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), pGBT9 (Clontech, Palo Alto, Calif.), pGAD10 (Clontech, PaloAlto, Calif.), pYADE4 and pYGAE2 and pYPGE2 (Brunelli and Pall, (1993)Yeast 9:1299-1308), pYPGE15 (Brunelli and Pall, (1993) Yeast9:1309-1318), pACTII (Dr. S. E. Elledge, Baylor College of Medicine),and picZ (InVitrogen Corp, San Diego, Calif.).

[0186] Alternatively, CARD-12 can be expressed in insect cells usingbaculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., Sf 9 cells)include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165)and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

[0187] In yet another embodiment, a nucleic acid of the invention isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed (1987)Nature 329:840), pCI (Promega), and pMT2PC (Kaufman et al. (1987) EMBOJ. 6:187-195). When used in mammalian cells, the expression vector'scontrol functions are often provided by viral regulatory elements. Forexample, commonly used promoters are derived from polyoma, Adenovirus 2,cytomegalovirus and Simian Virus 40. For other suitable expressionsystems for both prokaryotic and eukaryotic cells see chapters 16 and 17of Sambrook et al. (supra).

[0188] In another embodiment, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid).Tissue-specific regulatory elements are known in the art. Non-limitingexamples of suitable tissue-specific promoters include the albuminpromoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277),lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol.43:235-275), in particular promoters of T cell receptors (Winoto andBaltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al.(1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477),pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for examplethe murine hox promoters (Kessel and Gruss (1990) Science 249:374-379)and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev.3:537-546).

[0189] The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to CARD-12 mRNA. Regulatory sequences operativelylinked to a nucleic acid cloned in the antisense orientation can bechosen which direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub et al.(Reviews—Trends in Genetics, Vol. 1(1) 1986).

[0190] Another aspect of the invention pertains to host cells into whicha recombinant expression vector of the invention or isolated nucleicacid molecule of the invention has been introduced. The terms “hostcell” and “recombinant host cell” are used interchangeably herein. It isunderstood that such terms refer not only to the particular subject cellbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

[0191] A host cell can be any prokaryotic or eukaryotic cell. Forexample, CARD-12 protein can be expressed in bacterial cells such as E.coli, insect cells, yeast or mammalian cells (such as Chinese hamsterovary cells (CHO) or COS cells). Other suitable host cells are known tothose skilled in the art.

[0192] Vector DNA or an isolated nucleic acid molecule of the inventioncan be introduced into prokaryotic or eukaryotic cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook, et al. (supra), and other laboratorymanuals.

[0193] For stable transfection of mammalian cells, it is known that,depending upon the expression vector and transfection technique used,only a small fraction of cells may integrate the foreign DNA into theirgenome. In some cases vector DNA is retained by the host cell. In othercases the host cell does not retain vector DNA and retains only anisolated nucleic acid molecule of the invention carried by the vector.In some cases, and isolated nucleic acid molecule of the invention isused to transform a cell without the use of a vector.

[0194] In order to identify and select these integrants, a gene thatencodes a selectable marker (e.g., resistance to antibiotics) isgenerally introduced into the host cells along with the gene ofinterest. Preferred selectable markers include those which conferresistance to drugs, such as G418, hygromycin and methotrexate. Nucleicacid encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding CARD-12 or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

[0195] A host cell of the invention, such as a prokaryotic or eukaryotichost cell in culture, can be used to produce (i.e., express) a CARD-12protein. Accordingly, the invention further provides methods forproducing CARD-12 protein using the host cells of the invention. In oneembodiment, the method comprises culturing the host cell of theinvention (into which a recombinant expression vector or isolatednucleic acid molecule encoding CARD-12 has been introduced) in asuitable medium such that CARD-12 protein is produced. In anotherembodiment, the method further comprises isolating CARD-12 from themedium or the host cell.

[0196] CARD-12 nucleic acid molecules can be used in viral gene deliverysystems for gene therapy, e.g., adenoviral or retroviral gene deliverysystems.

[0197] CARD-12 nucleic acid molecules can also be used in non-viral genedelivery systems for gene therapy. Thus, another aspect of the inventionpertains to non-viral gene delivery systems, such as plasmid-based genedelivery systems. Non-viral gene delivery systems are described indetail by Huang et al. ((1999) Nonviral Vectors for Gene Therapy,Academic Press, San Diego, Calif.). Nonviral vectors have severalpotential advantages over their viral counterparts, including: reducedimmunogenicity; low acute toxicity; simplicity; and ease of large scaleproduction. Nonviral vectors can be delivered as naked DNA, bybioballistic bombardment, and in various complexes, includingliposome/DNA complexes (lipoplexes), polymer/DNA complexes (polyplexes),and liposome/polymer/DNA complexes (lipopolyplexes). Nonviral vectorsmay be administered by various routes, e.g., intravenous injection,peritoneal injection, intramuscular injection, subcutaneous injection,intratracheal injection, and aerosolization.

[0198] Naked DNA (i.e. free from association with, e.g.,transfection-facilitating proteins, viral particles, liposomalformulations, charged lipids and calcium phosphate precipitating), canbe expressed at its injection site or at a remote site. For example,naked DNA can be injected directly into skeletal muscle, liver, heartmuscle, and tumor tissue. For systemic administration, plasmid DNA mayneed to be protected from degradation by endonucleases during deliveryfrom the site of administration to the site of gene expression.

[0199] Bioballistic bombardment, also known as gene gun, allows for thepenetration of target cells in vitro, ex vivo, or in vivo. In thistechnique, DNA-coated gold particles are accelerated to a high velocityby an electric arc generated by a high voltage discharge. The method iseffective for a variety of organ types, including skin, liver, muscle,spleen, and pancreas. The gene gun transfer method is not dependent uponspecific cell surface receptors, cell cycle status, or the size of theDNA vector. Useful gene gun devices include the Accell® (PowderJectVaccines, Inc.) and the Helios™ (Bio-Rad). These devices create acompressed shock wave of helium gas, accelerating DNA-coated gold (ortungsten) particles to high speed, whereby the particles have sufficientmomentum to penetrate a target tissue.

[0200] Lipoplexes are typically made up of three components: a cationiclipid, a neutral colipid, and plasmid DNA that encodes one or more genesof interest. Commonly used cationic lipids include DOTMA, DMRIE,DC-chol, DOTAP, DMRIE, DDAB, DODAB/C, DOGS, DOSPA, SAINT-n, DOSPER,DPPES, DORIE, GAP-DLRIE, and DOTIM. Dioleoyl (DO) and dimyristoyl (DM)chains are thought to be especially effective for gene delivery.Cationic lipids are typically composed of a positively chargedheadgroup, a hydrophobic lipid anchor, and a linker that connects theheadgroup and anchor. Catioinc lipids used in lipoplexes can be dividedinto two broad classes: those that use cholesterol as the lipid anchorand those that use diacyl chains of varying lengths and extent ofsaturation. The number of protonatable amines on the headgroup mayaffect transfection activity, with multivalent headgroups beinggenerally more active than monovalent headgroups. The linker can be madeof a variety of chemical structures, e.g., ether, amide, carbamate,amine, urea, ester, and peptide bonds. Neutral colipids of lipoplexescommonly include DOPE, DOPC, and cholesterol. Generally, DOPE is used asthe neutral colipid with catioinc lipids that are based on cholesterol(e.g., DC-chol, GL-67) and cholesterol is used as the neutral colipidwith cationic lipids that harbor diacyl chains as the hydrophobic anchor(e.g., DOTAP, DOTIM).

[0201] Polyplexes are formed when cationic polymers are mixed with DNA.Cationic polymers used to from polyplexes are of two general types:linear polymers such as polylysine and spermine; and the branched chain,spherical, or globular polycations such as polyethyleneimine anddendrimers. Lipopolyplexes are formed by the incorporation of polylysineinto a lipoplex to form ternary complexes. DNA can be complexed with anatural biopolymer, e.g., gelatin or chitosan, functioning as a genecarrier to form nanospheres. Such biodegradable nanospheres have severaladvantages, including the coencapsulation of bioactive agents, e.g.nucleic acids and drugs, and the sustained release of the DNA.Gelatin-DNA or chitosan-DNA nanospheres are synthesized by mixing theDNA solution with an aqueous solution of gelatin or chitosan.

[0202] The effectiveness nonviral vectors may be enhanced by conjugationto ligands that direct the vector either to a particular cell type or toa particular location within a cell. Antibodies and other site-specificproteins can be attached to a vector, e.g., on the surface of the vectoror incorporated in the membrane. Following injection, these vectors bindefficiently and specifically to a target site. With respect toliposomes, ligands to a cell surface receptor can be incorporated intothe surface of a liposome by covalently modifying the ligand with alipid group and adding it during the formation of liposomes. Thefollowing classes of ligands can be incorporated into the nonviral DNAdelivery complexes of the invention in order to make them more effectivefor gene delivery: (1) peptides, e.g., peptides having a specific cellsurface receptor so that complexes will be targeted to specific cellsbearing the receptor; (2) nuclear localization signals, e.g., to promoteefficient entry of DNA into the nucleus; (3) pH-sensitive ligands, toencourage endosomal escape; (4) steric stabilizing agents, to preventdestabilization of the complexes after introduction into the biologicalmilieu. Gene chemistry approaches, e.g. peptide nucleic acids, can beused to couple ligands to DNA to improve the in vivo bioavailability andexpression of the DNA.

[0203] In plasmid-based, non-viral gene delivery systems it is oftenuseful to link a polypeptide (e.g., an antibody), nucleic acid molecule,or other compound to the gene delivery plasmid such that thepolypeptide, nucleic acid molecule or other compound remains associatedwith the plasmid following intracellular delivery in a manner that doesnot interfere with the transcriptional activity of the plasmid. This canbe accomplished using an appropriate biotin-conjugated peptide nucleicacid (PNA) clamp. A sequence complementary to the biotin-conjugated PNAclamp is inserted into the gene delivery plasmid. The biotin-conjugatedPNA will bind essentially irreversibly to the complementary sequenceinserted into the plasmid. A polypeptide, nucleic acid molecule or othercompound of interest can be conjugated to streptavidin. The streptavidinconjugate can bind to the biotin-PNA clamp bound to the plasmid. In thismanner, a polypeptide, nucleic acid molecule or other compound can bebound to a gene delivery plasmid such that the polypeptide, nucleic acidmolecule or other compound remains bound to the plasmid even within acell. Importantly, the PNA clamp-binding site in the plasmid must bechosen so as not to interfere with a needed promoter/enhancer or codingregion or otherwise disrupt the expression of the gene in the plasmid.An alternative approach employs a maleimide-conjugated PNA clamp.Polypeptides, nucleic acid molecules and other compounds containing afree thiol residue may be conjugated directly to the maleimide-PNA-DNAhybrid. As with the biotin-conjugated method, this conjugation does notdisturb the transcriptional activity of the plasmid if the PNA-bindingsite is chosen to be in a region of the plasmid not essential for geneactivity. Both of these approaches are described in detail by Zelphatiet al. ((2000) BioTechniques 28:304-315).

[0204] The host cells of the invention can also be used to producenonhuman transgenic animals. For example, in one embodiment, a host cellof the invention is a fertilized oocyte or an embryonic stem cell intowhich CARD-12-coding sequences have been introduced. Such host cells canthen be used to create non-human transgenic animals in which exogenousCARD-12 sequences have been introduced into their genome or homologousrecombinant animals in which endogenous CARD-12 sequences have beenaltered. Such animals are useful for studying the function and/oractivity of CARD-12 and for identifying and/or evaluating modulators ofCARD-12 activity. As used herein, a “transgenic animal” is a non-humananimal, preferably a mammal, more preferably a rodent such as a rat ormouse, in which one or more of the cells of the animal includes atransgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, etc. Atransgene is exogenous DNA which is integrated into the genome of a cellfrom which a transgenic animal develops and which remains in the genomeof the mature animal, thereby directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal. As used herein, an “homologous recombinant animal” is anon-human animal, preferably a mammal, more preferably a mouse, in whichan endogenous CARD-12 gene has been altered by homologous recombinationbetween the endogenous gene and an exogenous DNA molecule introducedinto a cell of the animal, e.g., an embryonic cell of the animal, priorto development of the animal.

[0205] A transgenic animal of the invention can be created byintroducing CARD-12-encoding nucleic acid into the male pronuclei of afertilized oocyte, e.g., by microinjection, retroviral infection, andallowing the oocyte to develop in a pseudopregnant female foster animal.The CARD-12 cDNA sequence, e.g., that of SEQ ID NO:1, SEQ ID NO:3 or thecDNA of ATCC can be introduced as a transgene into the genome of anon-human animal. Alternatively, a nonhuman homolog or ortholog of thehuman CARD-12 gene, such as a mouse CARD-12 gene, can be isolated basedon hybridization to the human CARD-12 cDNA and used as a transgene.Intronic sequences and polyadenylation signals can also be included inthe transgene to increase the efficiency of expression of the transgene.A tissue-specific regulatory sequence(s) can be operably linked to theCARD-12 transgene to direct expression of CARD-12 protein to particularcells. Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, U.S. Pat. No. 4,873,191 and in Hogan,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986). Similar methods are used for productionof other transgenic animals. A transgenic founder animal can beidentified based upon the presence of the CARD-12 transgene in itsgenome and/or expression of CARD-12 mRNA in tissues or cells of theanimals. A transgenic founder animal can then be used to breedadditional animals carrying the transgene. Moreover, transgenic animalscarrying a transgene encoding CARD-12 can further be bred to othertransgenic animals carrying other transgenes.

[0206] To create an homologous recombinant animal, a vector is preparedwhich contains at least a portion of a CARD-12 gene (e.g., a human or anon-human homolog of the CARD-12 gene, e.g., a murine CARD-12 gene) intowhich a deletion, addition or substitution has been introduced tothereby alter, e.g., functionally disrupt, the CARD-12 gene. In anembodiment, the vector is designed such that, upon homologousrecombination, the endogenous CARD-12 gene is functionally disrupted(i.e., no longer encodes a functional protein; also referred to as a“knock out” vector). Alternatively, the vector can be designed suchthat, upon homologous recombination, the endogenous CARD-12 gene ismutated or otherwise altered but still encodes functional protein (e.g.,the upstream regulatory region can be altered to thereby alter theexpression of the endogenous CARD-12 protein). In the homologousrecombination vector, the altered portion of the CARD-12 gene is flankedat its 5′ and 3′ ends by additional nucleic acid of the CARD-12 gene toallow for homologous recombination to occur between the exogenousCARD-12 gene carried by the vector and an endogenous CARD-12 gene in anembryonic stem cell. The additional flanking CARD-12 nucleic acid is ofsufficient length for successful homologous recombination with theendogenous gene. Typically, several kilobases of flanking DNA (both atthe 5′ and 3′ ends) are included in the vector (see, e.g., Thomas andCapecchi (1987) Cell 51:503 for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedCARD-12 gene has homologously recombined with the endogenous CARD-12gene are selected (see, e.g., Li et al. (1992) Cell 69:915). Theselected cells are then injected into a blastocyst of an animal (e.g., amouse) to form aggregation chimeras (see, e.g., Bradley inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach,Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo canthen be implanted into a suitable pseudopregnant female foster animaland the embryo brought to term. Progeny harboring the homologouslyrecombined DNA in their germ cells can be used to breed animals in whichall cells of the animal contain the homologously recombined DNA bygermline transmission of the transgene. Methods for constructinghomologous recombination vectors and homologous recombinant animals aredescribed further in Bradley (1991) Current Opinion in Bio/Technology2:823-829 and in PCT Publication Nos. WO 90/11354, WO 91/01140, WO92/0968, and WO 93/04169.

[0207] In another embodiment, transgenic non-humans animals can beproduced which contain selected systems which allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. (1992) Proc.Natl. Acad. Sci. USA 89:6232-6236. Another example of a recombinasesystem is the FLP recombinase system of Saccharomyces cerevisiae(O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP recombinasesystem is used to regulate expression of the transgene, animalscontaining transgenes encoding both the Cre recombinase and a selectedprotein are required. Such animals can be provided through theconstruction of “double” transgenic animals, e.g., by mating twotransgenic animals, one containing a transgene encoding a selectedprotein and the other containing a transgene encoding a recombinase.

[0208] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut et al.(1997) Nature 385:810-813 and PCT Publication Nos. WO 97/07668 and WO97/07669. In brief, a cell, e.g., a somatic cell, from the transgenicanimal can be isolated and induced to exit the growth cycle and enter Gophase. The quiescent cell can then be fused, e.g., through the use ofelectrical pulses, to an enucleated oocyte from an animal of the samespecies from which the quiescent cell is isolated. The reconstructedoocyte is then cultured such that it develops to morula or blastocyteand then transferred to pseudopregnant female foster animal. Theoffspring borne of this female foster animal will be a clone of theanimal from which the cell, e.g., the somatic cell, is isolated.

[0209] V. Pharmaceutical Compositions

[0210] The CARD-12 nucleic acid molecules, CARD-12 proteins, andanti-CARD-12 antibodies (also referred to herein as “active compounds”)of the invention can be incorporated into pharmaceutical compositionssuitable for administration. Such compositions typically comprise thenucleic acid molecule, protein, or antibody and a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

[0211] The invention includes methods for preparing pharmaceuticalcompositions for modulating the expression or activity of a polypeptideor nucleic acid of the invention. Such methods comprise formulating apharmaceutically acceptable carrier with an agent which modulatesexpression or activity of a polypeptide or nucleic acid of theinvention. Such compositions can further include additional activeagents. Thus, the invention further includes methods for preparing apharmaceutical composition by formulating a pharmaceutically acceptablecarrier with an agent which modulates expression or activity of apolypeptide or nucleic acid of the invention and one or more additionalactive compounds.

[0212] The agent which modulates expression or activity may, forexample, be a small molecule. For example, such small molecules includepeptides, peptidomimetics, amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e., including heteroorganicand organometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds. It is understood that appropriatedoses of small molecule agents depends upon a number of factors withinthe ken of the ordinarily skilled physician, veterinarian, orresearcher. The dose(s) of the small molecule will vary, for example,depending upon the identity, size, and condition of the subject orsample being treated, further depending upon the route by which thecomposition is to be administered, if applicable, and the effect whichthe practitioner desires the small molecule to have upon the nucleicacid or polypeptide of the invention. Exemplary doses include milligramor microgram amounts of the small molecule per kilogram of subject orsample weight (e.g., about 1 microgram per kilogram to about 500milligrams per kilogram, about 100 micrograms per kilogram to about 5milligrams per kilogram, or about 1 microgram per kilogram to about 50micrograms per kilogram. It is furthermore understood that appropriatedoses of a small molecule depend upon the potency of the small moleculewith respect to the expression or activity to be modulated. Suchappropriate doses may be determined using the assays described herein.When one or more of these small molecules is to be administered to ananimal (e.g., a human) in order to modulate expression or activity of apolypeptide or nucleic acid of the invention, a physician, veterinarian,or researcher may, for example, prescribe a relatively low dose atfirst, subsequently increasing the dose until an appropriate response isobtained. In addition, it is understood that the specific dose level forany particular subject will depend upon a variety of factors includingthe activity of the specific compound employed, the age, body weight,general health, gender, and diet of the subject, the time ofadministration, the route of administration, the rate of excretion, anydrug combination, and the degree of expression or activity to bemodulated.

[0213] A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as CARD-5orbic acid orsodium bisulfite; chelating agents such as ethylenediaminetetraaceticacid; buffers such as acetates, citrates or phosphates and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

[0214] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL (BASF; Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, CARD-5orbic acid, thimerosal, and the like. Inmany cases, it will be preferable to include isotonic agents, forexample, sugars, polyalcohols such as mannitol, sorbitol, sodiumchloride in the composition. Prolonged absorption of the injectablecompositions can be brought about by including in the composition anagent which delays absorption, for example, aluminum monostearate andgelatin.

[0215] Sterile injectable solutions can be prepared by incorporating theactive compound (e.g., a CARD-12 protein or anti-CARD-12 antibody) inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

[0216] Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring. For administrationby inhalation, the compounds are delivered in the form of an aerosolspray from pressured container or dispenser which contains a suitablepropellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0217] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0218] The compounds can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

[0219] In one embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

[0220] It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

[0221] For antibodies, the preferred dosage is 0.1 mg/kg to 100 mg/kg ofbody weight (generally 10 mg/kg to 20 mg/kg). If the antibody is to actin the brain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thebrain). A method for lipidation of antibodies is described by Cruikshanket al. ((1997) J. Acquired Immune Deficiency Syndromes and HumanRetrovirology 14:193).

[0222] The nucleic acid molecules of the invention can be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (U.S. Pat. No. 5,328,470) or by stereotactic injection(see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).The pharmaceutical preparation of the gene therapy vector can includethe gene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g. retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

[0223] The gene therapy vectors of the invention can be either viral ornon-viral. Examples of plasmid-based, non-viral vectors are discussed inHuang et al. (1999) Nonviral Vectors for Gene Therapy (supra). Amodified plasmid is one example of a non-viral gene delivery system.Peptides, proteins (including antibodies), and oligonucleotides may bestably conjugated to plasmid DNA by methods that do not interfere withthe transcriptional activity of the plasmid (Zelphati et al. (2000)BioTechniques 28:304-315). The attachment of proteins and/oroligonucleotides may influence the delivery and trafficking of theplasmid and thus render it a more effective pharmaceutical composition.

[0224] The pharmaceutical compositions can be included in a container,pack, or dispenser together with instructions for administration.

[0225] VI. Uses and Methods of the Invention

[0226] The nucleic acid molecules, proteins, protein homologues, andantibodies described herein can be used in one or more of the followingmethods: a) screening assays; b) detection assays (e.g., chromosomalmapping, tissue typing, forensic biology), c) predictive medicine (e.g.,diagnostic assays, prognostic assays, monitoring clinical trials, andpharmacogenomics); and d) methods of treatment (e.g., therapeutic andprophylactic). A CARD-12 protein interacts with other cellular proteinsand can thus be used for (i) regulation of cellular proliferation; (ii)regulation of cellular differentiation; and (iii) regulation of cellsurvival. The isolated nucleic acid molecules of the invention can beused to express CARD-12 protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect CARD-12mRNA (e.g., in a biological sample) or a genetic lesion in a CARD-12gene, and to modulate CARD-12 activity. In addition, the CARD-12proteins can be used to screen drugs or compounds which modulate theCARD-12 activity or expression as well as to treat disorderscharacterized by insufficient or excessive production of CARD-12 proteinor production of CARD-12 protein forms which have decreased or aberrantactivity compared to CARD-12 wild type protein. In addition, theanti-CARD-12 antibodies of the invention can be used to detect andisolate CARD-12 proteins and modulate CARD-12 activity.

[0227] This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

[0228] A. Screening Assays

[0229] The invention provides a method (also referred to herein as a“screening assay”) for identifying modulators, i.e., candidate or testcompounds or agents (e.g., peptides, peptidomimetics, small molecules orother drugs) which bind to CARD-12 proteins or biologically activeportions thereof or have a stimulatory or inhibitory effect on, forexample, CARD-12 expression or CARD-12 activity. An example of abiologically active portion of human CARD-12 is amino acids 139-227encoding a CARD domain.

[0230] Among the screening assays provided by the invention arescreening to identify molecules that prevent the dimerization ofCARD-12, screening to identify molecules which block the binding of aCARD containing polypeptide to CARD-12, and screening to identify acompetitive inhibitor of the binding of a nucleotide to the nucleotidebinding site of CARD-12. Screening assays, e.g., dimerization assays,can employ full-length CARD-12 or a portion of CARD-12, e.g., the CARDdomain, the nucleotide binding site domain, or the NAIP homology domain.

[0231] In one embodiment, the invention provides assays for screeningcandidate or test compounds which bind to or modulate the activity of aCARD-12 proteins or polypeptides or biologically active portionsthereof. The test compounds of the present invention can be obtainedusing any of the numerous approaches in combinatorial library methodsknown in the art, including: biological libraries; spatially addressableparallel solid phase or solution phase libraries; synthetic librarymethods requiring deconvolution; the “one-bead one-compound” librarymethod; and synthetic library methods using affinity chromatographyselection. The biological library approach is limited to peptidelibraries, while the other four approaches are applicable to peptide,non-peptide oligomer or small molecule libraries of compounds (Lam(1997) Anticancer Drug Des. 12:145). Examples of methods for thesynthesis of molecular libraries can be found in the art, for examplein: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb etal. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al.(1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303;Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al.(1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J.Med. Chem. 37:1233.

[0232] Libraries of compounds may be presented in solution (e.g.,Houghten (1992) Bio/Techniques 13:412-421), or on beads (Lam (1991)Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria(U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484;and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390;Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl.Acad. Sci. 87:6378-6382; and Felici (1991) J. Mol. Biol. 222:301-310).

[0233] Determining the ability of the test compound to modulate theactivity of CARD-12 or a biologically active portion thereof can beaccomplished, for example, by determining the ability of the CARD-12protein to bind to or interact with a CARD-12 target molecule. As usedherein, a “target molecule” is a molecule with which a CARD-12 proteinbinds or interacts in nature, for example, a molecule associated withthe internal surface of a cell membrane or a cytoplasmic molecule. ACARD-12 target molecule can be a non-CARD-12 molecule or a CARD-12protein or polypeptide of the present invention. In one embodiment, aCARD-12 target molecule is a component of an apoptotic signaltransduction pathway. The target, for example, can be a secondintracellular protein which has catalytic activity or a protein whichfacilitates the association of downstream signaling molecules withCARD-12.

[0234] Determining the ability of the test compound to modulate theactivity of CARD-12 or a biologically active portion thereof can beaccomplished, for example, by determining the ability of the CARD-12protein to bind to or interact with any of the specific proteins listedin the previous paragraph as CARD-12 target molecules. In anotherembodiment, CARD-12 target molecules include all proteins that bind to aCARD-12 protein or a fragment thereof in a two-hybrid system bindingassay which can be used without undue experimentation to isolate suchproteins from cDNA or genomic two-hybrid system libraries. The bindingassays described in this section can be cell-based or cell free(described subsequently).

[0235] Determining the ability of the CARD-12 protein to bind to orinteract with a CARD-12 target molecule can be accomplished by one ofthe methods described above for determining direct binding. In anembodiment, determining the ability of the CARD-12 protein to bind to orinteract with a CARD-12 target molecule can be accomplished bydetermining the activity of the target molecule. For example, theactivity of the target molecule can be determined by detecting inductionof a cellular second messenger of the target (e.g., intracellular Ca2+,diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity ofthe target on an appropriate substrate, detecting the induction of areporter gene (e.g., a CARD-12-responsive regulatory element operativelylinked to a nucleic acid encoding a detectable marker, e.g. luciferase),or detecting a cellular response, for example, cell survival, cellulardifferentiation, or cell proliferation. In addition, and in anotherembodiment, genes induced by CARD-12 expression can be identified byexpressing CARD-12 in a cell line and conducting a transcriptionalprofiling experiment wherein the mRNA expression patterns of the cellline transformed with an empty expression vector and the cell linetransformed with a CARD-12 expression vector are compared. The promotersof genes induced by CARD-12 expression can be operatively linked toreporter genes suitable for screening such as luciferase, secretedalkaline phosphatase, or beta-galactosidase and the resulting constructscould be introduced into appropriate expression vectors. A recombinantcell line containing CARD-12 and transfected with an expression vectorcontaining a CARD-12 responsive promoter operatively linked to areporter gene can be used to identify test compounds that modulateCARD-12 activity by assaying the expression of the reporter gene inresponse to contacting the recombinant cell line with test compounds.CARD-12 agonists can be identified as increasing the expression of thereporter gene and CARD-12 antagonists can be identified as decreasingthe expression of the reporter gene.

[0236] In another embodiment of the invention, the ability of a testcompound to modulate the activity of CARD-12, or biologically activeportions thereof can be determined by assaying the ability of the testcompound to modulate CARD-12-dependent pathways or processes where theCARD-12 target proteins that mediate the CARD-12 effect are known orunknown. Potential CARD-12-dependent pathways or processes include, butare not limited to, the modulation of cellular signal transductionpathways and their related second messenger molecules (e.g.,intracellular Ca2+, diacylglycerol, IP3, cAMP etc.), cellular enzymaticactivities, cellular responses (e.g., cell survival, cellulardifferentiation, or cell proliferation), or the induction or repressionof cellular or heterologous mRNAs or proteins. CARD-12-dependentpathways or processes could be assayed by standard cell-based or cellfree assays appropriate for the specific pathway or process under study.

[0237] In yet another embodiment, an assay of the present invention is acell-free assay comprising contacting a CARD-12 protein or biologicallyactive portion thereof with a test compound and determining the abilityof the test compound to bind to the CARD-12 protein or biologicallyactive portion thereof. Binding of the test compound to the CARD-12protein can be determined either directly or indirectly as describedabove. In one embodiment, a competitive binding assay includescontacting the CARD-12 protein or biologically active portion thereofwith a compound known to bind CARD-12 to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with a CARD-12 protein, whereindetermining the ability of the test compound to interact with a CARD-12protein comprises determining the ability of the test compound topreferentially bind to CARD-12 or biologically active portion thereof ascompared to the known binding compound.

[0238] In another embodiment, an assay is a cell-free assay comprisingcontacting CARD-12 protein or biologically active portion thereof with atest compound and determining the ability of the test compound tomodulate (e.g., stimulate or inhibit) the activity of the CARD-12protein or biologically active portion thereof. Determining the abilityof the test compound to modulate the activity of CARD-12 can beaccomplished, for example, by determining the ability of the CARD-12protein to bind to a CARD-12 target molecule by one of the methodsdescribed above for determining direct binding. In an alternativeembodiment, determining the ability of the test compound to modulate theactivity of CARD-12 can be accomplished by determining the ability ofthe CARD-12 protein to further modulate a CARD-12 target molecule. Forexample, the catalytic/enzymatic activity of the target molecule on anappropriate substrate can be determined as previously described.

[0239] In yet another embodiment, the cell-free assay comprisescontacting the CARD-12 protein or biologically active portion thereofwith a known compound which binds CARD-12 to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with a CARD-12 protein, whereindetermining the ability of the test compound to interact with a CARD-12protein comprises determining the ability of the CARD-12 protein topreferentially bind to or modulate the activity of a CARD-12 targetmolecule. The cell-free assays of the present invention are amenable touse of either the soluble form or a membrane-associated form of CARD-12.A membrane-associated form of CARD-12 refers to CARD-12 that interactswith a membrane-bound target molecule. In the case of cell-free assayscomprising the membrane-associated form of CARD-12, it may be desirableto utilize a solubilizing agent such that the membrane-associated formof CARD-12 is maintained in solution. Examples of such solubilizingagents include non-ionic detergents such as n-octylglucoside,n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®,Isotridecypoly(ethylene glycol ether)n,3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

[0240] In more than one embodiment of the above assay methods of thepresent invention, it may be desirable to immobilize either CARD-12 orits target molecule to facilitate separation of complexed fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay. Binding of a test compound toCARD-12, or interaction of CARD-12 with a target molecule in thepresence and absence of a candidate compound, can be accomplished in anyvessel suitable for containing the reactants. Examples of such vesselsinclude microtitre plates, test tubes, and micro-centrifuge tubes. Inone embodiment, a fusion protein can be provided which adds a domainthat allows one or both of the proteins to be bound to a matrix. Forexample, glutathione-S-transferase/CARD-12 fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or CARD-12 protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level ofCARD-12 binding or activity determined using standard techniques. In analternative embodiment, MYC or HA epitope tag CARD-12 fusion proteins orMYC or HA epitope tag target fusion proteins can be adsorbed ontoanti-MYC or anti-HA antibody coated microbeads or onto anti-MYC oranti-HA antibody coated microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or CARD-12 protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level ofCARD-12 binding or activity determined using standard techniques.

[0241] Other techniques for immobilizing proteins on matrices can alsobe used in the screening assays of the invention. For example, CARD-12or its target molecule can be immobilized utilizing conjugation ofbiotin and streptavidin. Biotinylated CARD-12 target molecules can beprepared from biotin-NHS (N-hydroxy-succinimide) using techniques wellknown in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford,Ill.), and immobilized in the wells of streptavidin-coated 96 wellplates (Pierce Chemical). Alternatively, antibodies reactive withCARD-12 or target molecules but which do not interfere with binding ofthe protein to its target molecule can be derivatized to the wells ofthe plate, and unbound target or protein trapped in the wells byantibody conjugation. Methods for detecting such complexes, in additionto those described above for the GST-immobilized complexes and epitopetag immobilized complexes, include immunodetection of complexes usingantibodies reactive with the CARD-12 or target molecule, as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the CARD-12 or a target molecule.

[0242] In another embodiment, modulators of CARD-12 expression areidentified in a method in which a cell is contacted with a candidatecompound and the expression of the CARD-12 promoter, mRNA or protein inthe cell is determined. The level of expression of CARD-12 mRNA orprotein in the presence of the candidate compound is compared to thelevel of expression of CARD-12 mRNA or protein in the absence of thecandidate compound. The candidate compound can then be identified as amodulator of CARD-12 expression based on this comparison. For example,when expression of CARD-12 mRNA or protein is greater (statisticallysignificantly greater) in the presence of the candidate compound than inits absence, the candidate compound is identified as a stimulator ofCARD-12 mRNA or protein expression. Alternatively, when expression ofCARD-12 mRNA or protein is less (statistically significantly less) inthe presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of CARD-12 mRNA orprotein expression. The level of CARD-12 mRNA or protein expression inthe cells can be determined by methods described herein for detectingCARD-12 mRNA or protein. The activity of the CARD-12 promoter can beassayed by linking the CARD-12 promoter to a reporter gene such asluciferase, secreted alkaline phosphatase, or beta-galactosidase andintroducing the resulting construct into an appropriate vector,transfecting a host cell line, and measuring the activity of thereporter gene in response to test compounds.

[0243] In yet another aspect of the invention, the CARD-12 proteins canbe used as “bait proteins” in a two-hybrid assay or three hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Bio/Techniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and PCT Publication No. WO 94/10300), to identify otherproteins, which bind to or interact with CARD-12 (“CARD-12-bindingproteins” or “CARD-12-bp”) and modulate CARD-12 activity. SuchCARD-12-binding proteins are also likely to be involved in thepropagation of signals by the CARD-12 proteins as, for example, upstreamor downstream elements of the CARD-12 pathway.

[0244] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for CARD-12 is fusedto a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. If the “bait” and the “prey” proteinsare able to interact, in vivo, forming a CARD-12-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene which encodes the protein which interacts with CARD-12.

[0245] In an embodiment of the invention, the ability of a test compoundto modulate the activity of CARD-12, or a biologically active portionthereof can be determined by assaying the ability of the test compoundto block the binding of CARD-12 to its target proteins in a yeast ormammalian two-hybrid system assay. This assay could be automated forhigh throughput drug screening purposes. In another embodiment of theinvention, CARD-12 and a target protein could be configured in thereverse two-hybrid system (Vidal et al. (1996) Proc. Natl. Acad. Sci.USA 93:10321-6 and Vidal et al. (1996) Proc. Natl. Acad. Sci. USA93:10315-20) designed specifically for efficient drug screening. In thereverse two-hybrid system, inhibition of a CARD-12 physical interactionwith a target protein would result in induction of a reporter gene incontrast to the normal two-hybrid system where inhibition of CARD-12physical interaction with a target protein would lead to reporter generepression. The reverse two-hybrid system is preferred for drugscreening because reporter gene induction is more easily assayed thanreport gene repression.

[0246] Alternative embodiments of the invention are proteins found tophysically interact with proteins that bind to CARD-12. CARD-12interactors could be configured into two-hybrid system baits and used intwo-hybrid screens to identify additional members of the CARD-12pathway. The interactors of CARD-12 interactors identified in this waycould be useful targets for therapeutic intervention in CARD-12 relateddiseases and pathologies and an assay of their enzymatic or bindingactivity could be useful for the identification of test compounds thatmodulate CARD-12 activity.

[0247] This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

[0248] B. Detection Assays

[0249] Portions or fragments of the cDNA sequences identified herein(and the corresponding complete gene sequences) can be used in numerousways as polynucleotide reagents. For example, these sequences can beused to: (i) map their respective genes on a chromosome; and, thus,locate gene regions associated with genetic disease; (ii) identify anindividual from a minute biological sample (tissue typing); and (iii)aid in forensic identification of a biological sample. Theseapplications are described in the subsections below.

[0250] 1. Chromosome Mapping

[0251] Once the sequence (or a portion of the sequence) of a gene hasbeen isolated, this sequence can be used to map the location of the geneon a chromosome. Accordingly, CARD-12 nucleic acid molecules describedherein or fragments thereof, can be used to map the location of CARD-12genes on a chromosome. The mapping of the CARD-12 sequences tochromosomes is an important first step in correlating these sequenceswith genes associated with disease.

[0252] Briefly, CARD-12 genes can be mapped to chromosomes by preparingPCR primers (preferably 15-25 bp in length) from the CARD-12 sequences.Computer analysis of CARD-12 sequences can be used to rapidly selectprimers that do not span more than one exon in the genomic DNA, thuscomplicating the amplification process. These primers can then be usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the CARD-12 sequences will yield an amplified fragment.

[0253] Somatic cell hybrids are prepared by fusing somatic cells fromdifferent mammals (e.g., human and mouse cells). As hybrids of human andmouse cells grow and divide, they gradually lose human chromosomes inrandom order, but retain the mouse chromosomes. By using media in whichmouse cells cannot grow, because they lack a particular enzyme, buthuman cells can, the one human chromosome that contains the geneencoding the needed enzyme, will be retained. By using various media,panels of hybrid cell lines can be established. Each cell line in apanel contains either a single human chromosome or a small number ofhuman chromosomes, and a full set of mouse chromosomes, allowing easymapping of individual genes to specific human chromosomes. (D'Eustachioet al. (1983) Science 220:919-924). Somatic cell hybrids containing onlyfragments of human chromosomes can also be produced using humanchromosomes with translocations and deletions.

[0254] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular sequence to a particular chromosome. Three ormore sequences can be assigned per day using a single thermal cycler.Using the CARD-12 sequences to design oligonucleotide primers,sublocalization can be achieved with panels of fragments from specificchromosomes. Other mapping strategies which can similarly be used to mapa CARD-12 sequence to its chromosome include in situ hybridization(described in Fan et al. (1990) Proc. Natl. Acad. Sci. USA 87:6223-27),pre-screening with labeled flow-sorted chromosomes, and pre-selection byhybridization to chromosome specific cDNA libraries.

[0255] Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical likecolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al., (Human Chromosomes: A Manual ofBasic Techniques (Pergamon Press, New York, 1988)).

[0256] Reagents for chromosome mapping can be used individually to marka single chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

[0257] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. (Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man, available on-line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and disease, mapped to the same chromosomal region, canthen be identified through linkage analysis (co-inheritance ofphysically adjacent genes), described in, e.g., Egeland et al. (1987)Nature, 325:783-787.

[0258] Moreover, differences in the DNA sequences between individualsaffected and unaffected with a disease associated with the CARD-12 genecan be determined. If a mutation is observed in some or all of theaffected individuals but not in any unaffected individuals, then themutation is likely to be the causative agent of the particular disease.Comparison of affected and unaffected individuals generally involvesfirst looking for structural alterations in the chromosomes such asdeletions or translocations that are visible from chromosome spreads ordetectable using PCR based on that DNA sequence. Ultimately, completesequencing of genes from several individuals can be performed to confirmthe presence of a mutation and to distinguish mutations frompolymorphisms.

[0259] A CARD-12 polypeptide and fragments and sequences thereof andantibodies specific thereto can be used to map the location of the geneencoding the polypeptide on a chromosome. This mapping can be carriedout by specifically detecting the presence of the polypeptide in membersof a panel of somatic cell hybrids between cells of a first species ofanimal from which the protein originates and cells from a second speciesof animal and then determining which somatic cell hybrid(s) expressesthe polypeptide and noting the chromosome(s) from the first species ofanimal that it contains. For examples of this technique, see Pajunen etal. (1988) Cytogenet. Cell Genet. 47:37-41 and Van Keuren et al. (1986)Hum. Genet. 74:34-40. Alternatively, the presence of the CARD-12polypeptide in the somatic cell hybrids can be determined by assaying anactivity or property of the polypeptide, for example, enzymaticactivity, as described in Bordelon-Riser et al. (1979) Somatic CellGenetics 5:597-613 and Owerbach et al. (1978) Proc. Natl. Acad. Sci. USA75:5640-5644.

[0260] 2. Tissue Typing

[0261] The CARD-12 sequences of the present invention can also be usedto identify individuals from minute biological samples. The UnitedStates military, for example, is considering the use of restrictionfragment length polymorphism (RFLP) for identification of its personnel.In this technique, an individual's genomic DNA is digested with one ormore restriction enzymes, and probed on a Southern blot to yield uniquebands for identification. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful as additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

[0262] Furthermore, the sequences of the present invention can be usedto provide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the CARD-12 sequences described herein can be used toprepare two PCR primers from the 5′ and 3′ ends of the sequences. Theseprimers can then be used to amplify an individual's DNA and subsequentlysequence it.

[0263] Panels of corresponding DNA sequences from individuals, preparedin this manner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The CARD-12 sequences of the invention uniquely represent portions ofthe human genome. Allelic variation occurs to some degree in the codingregions of these sequences, and to a greater degree in the noncodingregions. It is estimated that allelic variation between individualhumans occurs with a frequency of about once per each 500 bases. Each ofthe sequences described herein can, to some degree, be used as astandard against which DNA from an individual can be compared foridentification purposes. Because greater numbers of polymorphisms occurin the noncoding regions, fewer sequences are necessary to differentiateindividuals. The noncoding sequences of SEQ ID NO:1 can comfortablyprovide positive individual identification with a panel of perhaps 10 to1,000 primers which each yield a noncoding amplified sequence of 100bases. If predicted coding sequences, such as those in SEQ ID NO:3 areused, a more appropriate number of primers for positive individualidentification would be 500-2,000.

[0264] If a panel of reagents from CARD-12 sequences described herein isused to generate a unique identification database for an individual,those same reagents can later be used to identify tissue from thatindividual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

[0265] 3. Use of Partial Sequences in Forensic Biology

[0266] DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

[0267] The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO:1 are particularlyappropriate for this use as greater numbers of polymorphisms occur inthe noncoding regions, making it easier to differentiate individualsusing this technique. Examples of polynucleotide reagents include theCARD-12 sequences or portions thereof, e.g., fragments derived from thenoncoding regions of SEQ ID NO:1 which have a length of at least 20 or30 bases.

[0268] The sequences described herein can further be used to providepolynucleotide reagents, e.g., labeled or labelable probes which can beused in, for example, an in situ hybridization technique, to identify aspecific tissue, e.g., brain tissue. This can be very useful in caseswhere a forensic pathologist is presented with a tissue of unknownorigin. Panels of such CARD-12 probes can be used to identify tissue byspecies and/or by organ type.

[0269] In a similar fashion, these reagents, e.g., CARD-12 primers orprobes can be used to screen tissue culture for contamination (i.e.,screen for the presence of a mixture of different types of cells in aculture).

[0270] C. Predictive Medicine

[0271] The present invention also pertains to the field of predictivemedicine in which diagnostic assays, prognostic assays,pharmacogenomics, and monitoring clinical trials are used for prognostic(predictive) purposes to thereby treat an individual prophylactically.Accordingly, one aspect of the present invention relates to diagnosticassays for determining CARD-12 protein and/or nucleic acid expression aswell as CARD-12 activity, in the context of a biological sample (e.g.,blood, serum, cells, tissue) to thereby determine whether an individualis afflicted with a disease or disorder, or is at risk of developing adisorder, associated with aberrant CARD-12 expression or activity. Theinvention also provides for prognostic (or predictive) assays fordetermining whether an individual is at risk of developing a disorderassociated with CARD-12 protein, nucleic acid expression or activity.For example, mutations in a CARD-12 gene can be assayed in a biologicalsample. Such assays can be used for prognostic or predictive purpose tothereby prophylactically treat an individual prior to the onset of adisorder characterized by or associated with CARD-12 protein, nucleicacid expression or activity.

[0272] Another aspect of the invention provides methods for determiningCARD-12 protein, nucleic acid expression or CARD-12 activity in anindividual to thereby select appropriate therapeutic or prophylacticagents for that individual (referred to herein as “pharmacogenomics”).Pharmacogenomics allows for the selection of agents (e.g., drugs) fortherapeutic or prophylactic treatment of an individual based on thegenotype of the individual (e.g., the genotype of the individualexamined to determine the ability of the individual to respond to aparticular agent.)

[0273] Yet another aspect of the invention pertains to monitoring theinfluence of agents (e.g., drugs or other compounds) on the expressionor activity of CARD-12 in clinical trials.

[0274] These and other agents are described in further detail in thefollowing sections.

[0275] 1. Diagnostic Assays

[0276] An exemplary method for detecting the presence or absence ofCARD-12 in a biological sample involves obtaining a biological samplefrom a test subject and contacting the biological sample with a compoundor an agent capable of detecting CARD-12 protein or nucleic acid (e.g.,mRNA, genomic DNA) that encodes CARD-12 protein such that the presenceof CARD-12 is detected in the biological sample. An agent for detectingCARD-12 mRNA or genomic DNA is a labeled nucleic acid probe capable ofhybridizing to CARD-12 mRNA or genomic DNA. The nucleic acid probe canbe, for example, a full-length CARD-12 nucleic acid, such as the nucleicacid of SEQ ID NO:1 or 3, or a portion thereof, such as anoligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides inlength and sufficient to specifically hybridize under stringentconditions to mRNA or genomic DNA. Other suitable probes for use in thediagnostic assays of the invention are described herein.

[0277] An agent for detecting CARD-12 protein can be an antibody capableof binding to CARD-12 protein, preferably an antibody with a detectablelabel. Antibodies can be polyclonal, or more preferably, monoclonal. Anintact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can beused. The term “labeled”, with regard to the probe or antibody, isintended to encompass direct labeling of the probe or antibody bycoupling (i.e., physically linking) a detectable substance to the probeor antibody, as well as indirect labeling of the probe or antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently labeled secondary antibody and end-labeling of a DNA probewith biotin such that it can be detected with fluorescently labeledstreptavidin. The term “biological sample” is intended to includetissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject. That is, thedetection method of the invention can be used to detect CARD-12 mRNA,protein, or genomic DNA in a biological sample in vitro as well as invivo. For example, in vitro techniques for detection of CARD-12 mRNAinclude Northern hybridizations and in situ hybridizations. In vitrotechniques for detection of CARD-12 protein include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence. In vitro techniques for detection of CARD-12 genomicDNA include Southern hybridizations. Furthermore, in vivo techniques fordetection of CARD-12 protein include introducing into a subject alabeled anti-CARD-12 antibody. For example, the antibody can be labeledwith a radioactive marker whose presence and location in a subject canbe detected by standard imaging techniques.

[0278] In one embodiment, the biological sample contains proteinmolecules from the test subject. Alternatively, the biological samplecan contain mRNA molecules from the test subject or genomic DNAmolecules from the test subject. A biological sample is a peripheralblood leukocyte sample isolated by conventional means from a subject.

[0279] In another embodiment, the methods further involve obtaining acontrol biological sample from a control subject, contacting the controlsample with a compound or agent capable of detecting CARD-12 protein,mRNA, or genomic DNA, such that the presence of CARD-12 protein, mRNA orgenomic DNA is detected in the biological sample, and comparing thepresence of CARD-12 protein, mRNA or genomic DNA in the control samplewith the presence of CARD-12 protein, mRNA or genomic DNA in the testsample.

[0280] The invention also encompasses kits for detecting the presence ofCARD-12 in a biological sample (a test sample). Such kits can be used todetermine if a subject is suffering from or is at increased risk ofdeveloping a disorder associated with aberrant expression of CARD-12(e.g., an immunological disorder). For example, the kit can comprise alabeled compound or agent capable of detecting CARD-12 protein or mRNAin a biological sample and means for determining the amount of CARD-12in the sample (e.g., an anti-CARD-12 antibody or an oligonucleotideprobe which binds to DNA encoding CARD-12, e.g., SEQ ID NO:1 or SEQ IDNO:3). Kits may also include instruction for observing that the testedsubject is suffering from or is at risk of developing a disorderassociated with aberrant expression of CARD-12 if the amount of CARD-12protein or mRNA is above or below a normal level.

[0281] For antibody-based kits, the kit may comprise, for example: (1) afirst antibody (e.g., attached to a solid support) which binds toCARD-12 protein; and, optionally, (2) a second, different antibody whichbinds to CARD-12 protein or the first antibody and is conjugated to adetectable agent. For oligonucleotide-based kits, the kit may comprise,for example: (1) a oligonucleotide, e.g., a detectably labelledoligonucleotide, which hybridizes to a CARD-12 nucleic acid sequence or(2) a pair of primers useful for amplifying a CARD-12 nucleic acidmolecule.

[0282] The kit may also comprise, e.g., a buffering agent, apreservative, or a protein stabilizing agent. The kit may also comprisecomponents necessary for detecting the detectable agent (e.g., an enzymeor a substrate). The kit may also contain a control sample or a seriesof control samples which can be assayed and compared to the test samplecontained. Each component of the kit is usually enclosed within anindividual container and all of the various containers are within asingle package along with instructions for observing whether the testedsubject is suffering from or is at risk of developing a disorderassociated with aberrant expression of CARD-12.

[0283] 2. Prognostic Assays

[0284] The methods described herein can furthermore be utilized asdiagnostic or prognostic assays to identify subjects having or at riskof developing a disease or disorder associated with aberrant CARD-12expression or activity. For example, the assays described herein, suchas the preceding diagnostic assays or the following assays, can beutilized to identify a subject having or at risk of developing adisorder associated with CARD-12 protein, nucleic acid expression oractivity. Alternatively, the prognostic assays can be utilized toidentify a subject having or at risk for developing such a disease ordisorder. Thus, the present invention provides a method in which a testsample is obtained from a subject and CARD-12 protein or nucleic acid(e.g., mRNA, genomic DNA) is detected, wherein the presence of CARD-12protein or nucleic acid is diagnostic for a subject having or at risk ofdeveloping a disease or disorder associated with aberrant CARD-12expression or activity. As used herein, a “test sample” refers to abiological sample obtained from a subject of interest. For example, atest sample can be a biological fluid (e.g., serum), cell sample, ortissue. Furthermore, the prognostic assays described herein can be usedto determine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant CARD-12 expression or activity. For example,such methods can be used to determine whether a subject can beeffectively treated with a specific agent or class of agents (e.g.,agents of a type which decrease CARD-12 activity). Thus, the presentinvention provides methods for determining whether a subject can beeffectively treated with an agent for a disorder associated withaberrant CARD-12 expression or activity in which a test sample isobtained and CARD-12 protein or nucleic acid is detected (e.g., whereinthe presence of CARD-12 protein or nucleic acid is diagnostic for asubject that can be administered the agent to treat a disorderassociated with aberrant CARD-12 expression or activity).

[0285] The methods of the invention can also be used to detect geneticlesions or mutations in a CARD-12 gene, thereby determining if a subjectwith the lesioned gene is at risk for a disorder characterized byaberrant cell proliferation and/or differentiation. In preferredembodiments, the methods include detecting, in a sample of cells fromthe subject, the presence or absence of a genetic lesion characterizedby at least one of an alteration affecting the integrity of a geneencoding a CARD-12-protein, or the mis-expression of the CARD-12 gene.For example, such genetic lesions can be detected by CARD-5ertaining theexistence of at least one of 1) a deletion of one or more nucleotidesfrom a CARD-12 gene; 2) an addition of one or more nucleotides to aCARD-12 gene; 3) a substitution of one or more nucleotides of a CARD-12gene; 4) a chromosomal rearrangement of a CARD-12 gene; 5) an alterationin the level of a messenger RNA transcript of a CARD-12 gene; 6)aberrant modification of a CARD-12 gene, such as of the methylationpattern of the genomic DNA; 7) the presence of a non-wild type splicingpattern of a messenger RNA transcript of a CARD-12 gene (e.g., caused bya mutation in a splice donor or splice acceptor site); 8) a non-wildtype level of a CARD-12-protein; 9) allelic loss of a CARD-12 gene; and10) inappropriate post-translational modification of a CARD-12-protein.As described herein, there are a large number of assay techniques knownin the art which can be used for detecting lesions in a CARD-12 gene. Abiological sample is a peripheral blood leukocyte sample isolated byconventional means from a subject.

[0286] In certain embodiments, detection of the lesion involves the useof a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the CARD-12 gene(see, e.g., Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). Thismethod can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a CARD-12 gene under conditionssuch that hybridization and amplification of the CARD-12-gene (ifpresent) occurs, and detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. It is anticipatedthat PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein.

[0287] Alternative amplification methods include: self sustainedsequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

[0288] In an alternative embodiment, mutations in a CARD-12 gene from asample cell can be identified by alterations in restriction enzymecleavage patterns. For example, sample and control DNA is isolated,amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined by gelelectrophoresis and compared. Differences in fragment length sizesbetween sample and control DNA indicates mutations in the sample DNA.Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Pat.No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

[0289] In other embodiments, genetic mutations in CARD-12 can beidentified by hybridizing a sample and control nucleic acids, e.g., DNAor RNA, to high density arrays containing hundreds or thousands ofoligonucleotides probes (Cronin et al. (1996) Human Mutation 7:244-255;Kozal et al. (1996) Nature Medicine 2:753-759). For example, geneticmutations in CARD-12 can be identified in two-dimensional arrayscontaining light-generated DNA probes as described in Cronin et al.supra. Briefly, a first hybridization array of probes can be used toscan through long stretches of DNA in a sample and control to identifybase changes between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

[0290] In yet another embodiment, any of a variety of sequencingreactions known in the art can be used to directly sequence the CARD-12gene and detect mutations by comparing the sequence of the sampleCARD-12 with the corresponding wild-type (control) sequence. Examples ofsequencing reactions include those based on techniques developed byMaxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplatedthat any of a variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays ((1995) Bio/Techniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT PublicationNo. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; andGriffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).

[0291] Other methods for detecting mutations in the CARD-12 gene includemethods in which protection from cleavage agents is used to detectmismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al.(1985) Science 230:1242). In general, the art technique of “mismatchcleavage” starts by providing heteroduplexes of formed by hybridizing(labeled) RNA or DNA containing the wild-type CARD-12 sequence withpotentially mutant RNA or DNA obtained from a tissue sample. Thedouble-stranded duplexes are treated with an agent which cleavessingle-stranded regions of the duplex such as which will exist due tobasepair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with S1 nuclease to enzymatically digesting the mismatchedregions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can betreated with hydroxylamine or osmium tetroxide and with piperidine inorder to digest mismatched regions. After digestion of the mismatchedregions, the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, e.g., Cottonet al (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al (1992)Methods Enzymol. 217:286-295. In an embodiment, the control DNA or RNAcan be labeled for detection.

[0292] In still another embodiment, the mismatch cleavage reactionemploys one or more proteins that recognize mismatched base pairs indouble-stranded DNA (so called “DNA mismatch repair” enzymes) in definedsystems for detecting and mapping point mutations in CARD-12 cDNAsobtained from samples of cells. For example, the mutY enzyme of E. colicleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLacells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis15:1657-1662). According to an exemplary embodiment, a probe based on aCARD-12 sequence, e.g., a wild-type CARD-12 sequence, is hybridized to acDNA or other DNA product from a test cell(s). The duplex is treatedwith a DNA mismatch repair enzyme, and the cleavage products, if any,can be detected from electrophoresis protocols or the like. See, e.g.,U.S. Pat. No. 5,459,039.

[0293] In other embodiments, alterations in electrophoretic mobilitywill be used to identify mutations in CARD-12 genes. For example, singlestrand conformation polymorphism (SSCP) may be used to detectdifferences in electrophoretic mobility between mutant and wild typenucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766,see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992) GenetAnal Tech Appl 9:73-79). Single-stranded DNA fragments of sample andcontrol CARD-12 nucleic acids will be denatured and allowed to renature.The secondary structure of single-stranded nucleic acids variesaccording to sequence, the resulting alteration in electrophoreticmobility enables the detection of even a single base change.

[0294] The DNA fragments may be labeled or detected with labeled probes.The sensitivity of the assay may be enhanced by using RNA (rather thanDNA), in which the secondary structure is more sensitive to a change insequence. In an embodiment, the subject method utilizes heteroduplexanalysis to separate double stranded heteroduplex molecules on the basisof changes in electrophoretic mobility (Keen et al. (1991) Trends Genet7:5).

[0295] In yet another embodiment, the movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (DGGE) (Myers etal. (1985) Nature 313:495). When DGGE is used as the method of analysis,DNA will be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

[0296] Examples of other techniques for detecting point mutationsinclude, but are not limited to, selective oligonucleotidehybridization, selective amplification, or selective primer extension.For example, oligonucleotide primers may be prepared in which the knownmutation is placed centrally and then hybridized to target DNA underconditions which permit hybridization only if a perfect match is found(Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. NatlAcad. Sci USA 86:6230). Such allele specific oligonucleotides arehybridized to PCR amplified target DNA or a number of differentmutations when the oligonucleotides are attached to the hybridizingmembrane and hybridized with labeled target DNA.

[0297] Alternatively, allele specific amplification technology whichdepends on selective PCR amplification may be used in conjunction withthe instant invention. Oligonucleotides used as primers for specificamplification may carry the mutation of interest in the center of themolecule (so that amplification depends on differential hybridization)(Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme3′ end of one primer where, under appropriate conditions, mismatch canprevent, or reduce polymerase extension (Prossner (1993) Tibtech11:238). In addition, it may be desirable to introduce a novelrestriction site in the region of the mutation to create cleavage-baseddetection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It isanticipated that in certain embodiments amplification may also beperformed using Taq ligase for amplification (Barany (1991) Proc. Natl.Acad. Sci USA 88:189). In such cases, ligation will occur only if thereis a perfect match at the 3′ end of the 5′ sequence making it possibleto detect the presence of a known mutation at a specific site by lookingfor the presence or absence of amplification.

[0298] The methods described herein may be performed, for example, byutilizing pre-packaged diagnostic kits comprising at least one probenucleic acid or antibody reagent described herein, which may beconveniently used, e.g., in clinical settings to diagnose patientsexhibiting symptoms or family history of a disease or illness involvinga CARD-12 gene.

[0299] Furthermore, any cell type or tissue, preferably peripheral bloodleukocytes, in which CARD-12 is expressed may be utilized in theprognostic assays described herein.

[0300] 3. Pharmacogenomics

[0301] Agents, or modulators which have a stimulatory or inhibitoryeffect on CARD-12 activity (e.g., CARD-12 gene expression) as identifiedby a screening assay described herein can be administered to individualsto treat (prophylactically or therapeutically) disorders (e.g., animmunological disorder) associated with aberrant CARD-12 activity. Inconjunction with such treatment, the pharmacogenomics (i.e., the studyof the relationship between an individual's genotype and thatindividual's response to a foreign compound or drug) of the individualmay be considered. Differences in metabolism of therapeutics can lead tosevere toxicity or therapeutic failure by altering the relation betweendose and blood concentration of the pharmacologically active drug. Thus,the pharmacogenomics of the individual permits the selection ofeffective agents (e.g., drugs) for prophylactic or therapeutictreatments based on a consideration of the individual's genotype. Suchpharmacogenomics can further be used to determine appropriate dosagesand therapeutic regimens. Accordingly, the activity of CARD-12 protein,expression of CARD-12 nucleic acid, or mutation content of CARD-12 genesin an individual can be determined to thereby select appropriateagent(s) for therapeutic or prophylactic treatment of the individual.

[0302] Pharmacogenomics deals with clinically significant hereditaryvariations in the response to drugs due to altered drug disposition andabnormal action in affected persons. See, e.g., Linder (1997) Clin.Chem. 43(2):254-266. In general, two types of pharmacogenetic conditionscan be differentiated. Genetic conditions transmitted as a single factoraltering the way drugs act on the body (altered drug action) or geneticconditions transmitted as single factors altering the way the body actson drugs (altered drug metabolism). These pharmacogenetic conditions canoccur either as rare defects or as polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[0303] As an illustrative embodiment, the activity of drug metabolizingenzymes is a major determinant of both the intensity and duration ofdrug action. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C 19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM exhibit no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so-called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

[0304] Thus, the activity of CARD-12 protein, expression of CARD-12nucleic acid, or mutation content of CARD-12 genes in an individual canbe determined to thereby select appropriate agent(s) for therapeutic orprophylactic treatment of the individual. In addition, pharmacogeneticstudies can be used to apply genotyping of polymorphic alleles encodingdrug-metabolizing enzymes to the identification of an individual's drugresponsiveness phenotype. This knowledge, when applied to dosing or drugselection, can avoid adverse reactions or therapeutic failure and thusenhance therapeutic or prophylactic efficiency when treating a subjectwith a CARD-12 modulator, such as a modulator identified by one of theexemplary screening assays described herein.

[0305] 4. Monitoring of Effects During Clinical Trials

[0306] Monitoring the influence of agents (e.g., drugs, compounds) onthe expression or activity of CARD-12 (e.g., the ability to modulateaberrant cell proliferation and/or differentiation) can be applied notonly in basic drug screening, but also in clinical trials. For example,the effectiveness of an agent determined by a screening assay asdescribed herein to increase CARD-12 gene expression, protein levels, orupregulate CARD-12 activity, can be monitored in clinical trails ofsubjects exhibiting decreased CARD-12 gene expression, protein levels,or downregulated CARD-12 activity. Alternatively, the effectiveness ofan agent determined by a screening assay to decrease CARD-12 geneexpression, protein levels, or downregulated CARD-12 activity, can bemonitored in clinical trials of subjects exhibiting increased CARD-12gene expression, protein levels, or upregulated CARD-12 activity. Insuch clinical trials, the expression or activity of CARD-12 and,preferably, other genes that have been implicated in, for example, acellular proliferation disorder can be used as a “read out” or markersof the immune responsiveness of a particular cell.

[0307] For example, and not by way of limitation, genes, includingCARD-12, that are modulated in cells by treatment with an agent (e.g.,compound, drug or small molecule) which modulates CARD-12 activity(e.g., identified in a screening assay as described herein) can beidentified. Thus, to study the effect of agents on cellularproliferation disorders, for example, in a clinical trial, cells can beisolated and RNA prepared and analyzed for the levels of expression ofCARD-12 and other genes implicated in the disorder. The levels of geneexpression (i.e., a gene expression pattern) can be quantified byNorthern blot analysis or RT-PCR, as described herein, or alternativelyby measuring the amount of protein produced, by one of the methods asdescribed herein, or by measuring the levels of activity of CARD-12 orother genes. In this way, the gene expression pattern can serve as amarker, indicative of the physiological response of the cells to theagent. Accordingly, this response state may be determined before, and atvarious points during, treatment of the individual with the agent.

[0308] In an embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) comprising the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of a CARD-12 protein,mRNA, or genomic DNA in the preadministration sample; (iii) obtainingone or more post-administration samples from the subject; (iv) detectingthe level of expression or activity of the CARD-12 protein, mRNA, orgenomic DNA in the post-administration samples; (v) comparing the levelof expression or activity of the CARD-12 protein, mRNA, or genomic DNAin the pre-administration sample with the CARD-12 protein, mRNA, orgenomic DNA in the post administration sample or samples; and (vi)altering the administration of the agent to the subject accordingly. Forexample, increased administration of the agent may be desirable toincrease the expression or activity of CARD-12 to higher levels thandetected, i.e., to increase the effectiveness of the agent.Alternatively, decreased administration of the agent may be desirable todecrease expression or activity of CARD-12 to lower levels thandetected, i.e., to decrease the effectiveness of the agent.

[0309] 5. Transcriptional Profiling

[0310] The CARD-12 nucleic acid molecules described herein, includingsmall oligonucleotides, can be used in transcriptionally profiling. Forexample, these nucleic acids can be used to examine the expression ofCARD-12 in normal tissue or cells and in tissue or cells subject to adisease state, e.g., tissue or cells derived from a patient having adisease of interest or cultured cells which model or reflect a diseasestate of interest, e.g., cells of a cultured tumor cell line. Bymeasuring expression of CARD-12, together or individually, a profile ofexpression in normal and disease states can be developed. This profilecan be used diagnostically and to examine the effectiveness of atherapeutic regime.

[0311] C. Methods of Treatment

[0312] The present invention provides for both prophylactic andtherapeutic methods of treating a subject at risk of (or susceptible to)a disorder or having a disorder associated with aberrant CARD-12expression or activity, examples of which are provided herein.

[0313] 1. Prophylactic Methods

[0314] In one aspect, the invention provides a method for preventing ina subject, a disease or condition associated with an aberrant CARD-12expression or activity, by administering to the subject an agent whichmodulates CARD-12 expression or at least one CARD-12 activity. Subjectsat risk for a disease which is caused or contributed to by aberrantCARD-12 expression or activity can be identified by, for example, any ora combination of diagnostic or prognostic assays as described herein.Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the CARD-12 aberrancy, suchthat a disease or disorder is prevented or, alternatively, delayed inits progression. Depending on the type of CARD-12 aberrancy, forexample, a CARD-12 agonist or CARD-12 antagonist agent can be used fortreating the subject. The appropriate agent can be determined based onscreening assays described herein.

[0315] 2. Therapeutic Methods

[0316] Another aspect of the invention pertains to methods of modulatingCARD-12 expression or activity for therapeutic purposes. The modulatorymethod of the invention involves contacting a cell with an agent thatmodulates one or more of the activities of CARD-12 protein activityassociated with the cell. An agent that modulates CARD-12 proteinactivity can be an agent as described herein, such as a nucleic acid ora protein, a naturally-occurring cognate ligand of a CARD-12 protein, apeptide, a CARD-12 peptidomimetic, or other small molecule. In oneembodiment, the agent stimulates one or more of the biologicalactivities of CARD-12 protein. Examples of such stimulatory agentsinclude active CARD-12 protein and a nucleic acid molecule encodingCARD-12 that has been introduced into the cell. In another embodiment,the agent inhibits one or more of the biological activities of CARD-12protein. Examples of such inhibitory agents include antisense CARD-12nucleic acid molecules and anti-CARD-12 antibodies. These modulatorymethods can be performed in vitro (e.g., by culturing the cell with theagent) or, alternatively, in vivo (e.g., by administering the agent to asubject). As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant expression or activity of a CARD-12 protein or nucleic acidmolecule or a disorder related to CARD-12 expression or activity. In oneembodiment, the method involves administering an agent (e.g., an agentidentified by a screening assay described herein), or combination ofagents that modulates (e.g., upregulates or downregulates) CARD-12expression or activity. In another embodiment, the method involvesadministering a CARD-12 protein or nucleic acid molecule as therapy tocompensate for reduced or aberrant CARD-12 expression or activity.

[0317] Stimulation of CARD-12 activity is desirable in situations inwhich CARD-12 is abnormally downregulated and/or in which increasedCARD-12 activity is likely to have a beneficial effect. Conversely,inhibition of CARD-12 activity is desirable in situations in whichCARD-12 is abnormally upregulated, e.g., in myocardial infarction,and/or in which decreased CARD-12 activity is likely to have abeneficial effect.

[0318] Equivalents

[0319] Those skilled in the art will recognize, or be able toCARD-5ertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. Such equivalents are intended to be encompassed by the followingclaims.

What is claimed is:
 1. An isolated nucleic acid molecule comprising atleast 50 contiguous nucleotides of SEQ ID NO:1.
 2. The isolated nucleicacid molecule of claim 1 wherein the nucleic molecule comprises at least150 contiguous nucleotide of SEQ ID NO:1.
 3. The isolated nucleic acidmolecule of claim 1 wherein the nucleic acid molecule comprises at least500 contiguous nucleotide of SEQ ID NO:1.
 4. An isolated nucleic acidmolecule comprising at least 50 contiguous nucleotides of SEQ ID NO:3.5. The isolated nucleic acid molecule of claim 4 wherein the nucleicacid molecule comprises at least 150 contiguous nucleotide of SEQ IDNO:3.
 6. The isolated nucleic acid molecule of claim 4 wherein thenucleic acid molecule comprises at least 500 contiguous nucleotide ofSEQ ID NO:3.
 7. The isolated nucleic acid molecule of claim 1 whereinthe nucleic acid molecule comprises the nucleotide sequence of SEQ IDNO:1.
 8. The isolated nucleic acid molecule of claim 1 wherein thenucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:3.9. An isolated nucleic acid molecule comprising a nucleotide sequenceencoding a polypeptide comprising at least 25 contiguous amino acids ofSEQ ID NO:2.
 10. The isolated nucleic acid molecule of claim 9 whereinthe polypeptide comprises at least 150 contiguous amino acids of SEQ IDNO:2.
 11. The isolated nucleic acid molecule of claim 9 wherein thepolypeptide comprises at least one domain selected from the groupconsisting of a CARD domain, a NACHT domain, a leucine rich repeatdomain, and an NBS domain.
 12. The isolated nucleic acid molecule ofclaim 9 wherein the polypeptide comprises at least two domains selectedfrom the group consisting of a CARD domain, a NACHT motif domain, aleucine rich repeat domain, and an NBS domain.
 13. The isolated nucleicacid molecule of claim 9 wherein the polypeptide comprises at least twoNACHT motif domains.
 14. The isolated nucleic acid molecule of claim 9wherein the polypeptide comprises at least two leucine rich repeatdomains.
 15. The isolated nucleic acid molecule of claim 9 wherein thepolypeptide comprises the amino acid sequence of SEQ ID NO:2.
 16. Anisolated nucleic acid molecule that hybridizes to a nucleic acidmolecule consisting of the nucleotide sequence of SEQ ID NO:3 underconditions of incubation at 45° C. in 6.0× SSC followed by washing in0.2× SSC/0.1% SDS at 65° C.
 17. The nucleic acid molecule of claim 1further comprising vector nucleic acid sequences.
 18. The nucleic acidmolecule of claim 17 wherein the vector nucleic acid sequences compriseexpression control sequences.
 19. A host cell that contains the nucleicacid molecule of claim
 1. 20. The host cell of claim 19 wherein the hostcell is a mammalian cell.
 21. An isolated polypeptide comprising atleast 15 contiguous amino acids of SEQ ID NO:2.
 22. The isolatedpolypeptide of claim 21 wherein the polypeptide comprises at least 75contiguous amino acids of SEQ ID NO:2.
 23. The isolated polypeptide ofclaim 21 comprising the amino acid sequence of SEQ ID NO:2.
 24. Anisolated polypeptide comprising the amino acid sequence of SEQ ID NO:2wherein up to 10 amino acids are replaced by conservative substitutions.25. A fusion protein comprising at least 25 contiguous amino acids ofSEQ ID NO:2
 26. An antibody which selectively binds to a polypeptideconsisting of the amino acid sequence of SEQ IN NO:2.
 27. A method fordetecting the presence of a polypeptide of claim 21 in a sample,comprising: a) contacting the sample with a compound which selectivelybinds to a polypeptide of claim 2; and b) determining whether thecompound binds to the polypeptide in the sample.
 28. The method of claim27, wherein the compound that binds to the polypeptide is an antibody.29. A kit comprising a compound that selectively binds to a polypeptideof claim 22 and instructions for use.
 30. A method for detecting thepresence of a nucleic acid molecule of claim 1 in a sample, comprisingthe steps of: a) contacting the sample with a nucleic acid probe orprimer which selectively hybridizes to the nucleic acid molecule ofclaim 1; and b) determining whether the nucleic acid probe or primerbinds to a nucleic acid molecule in the sample.
 31. The method of claim30, wherein the sample comprises mRNA molecules and is contacted with anucleic acid probe.
 32. A kit comprising a compound that selectivelyhybridizes to a nucleic acid molecule of claim 1 and instructions foruse.
 33. A method for identifying a compound which binds to apolypeptide of comprising the amino acid sequence of SEQ ID NO:2 themethod comprising the steps of: a) contacting a polypeptide, or a cellexpressing a polypeptide comprising the amino acid sequence of SEQ IDNO:2 with a test compound; and b) determining whether the polypeptidebinds to the test compound.
 34. The method of claim 33, wherein thebinding of the test compound to the polypeptide is detected by a methodselected from the group consisting of: a) detection of binding by directdetecting of test compound/polypeptide binding; b) detection of bindingusing a competition binding assay; c) detection of binding using anassay for CARD-12-mediated signal transduction; and d) detection ofbinding of a CARD-12 to a CARD domain.
 35. A method for identifying acompound that modulates the activity of a polypeptide comprising theamino acid sequence of SEQ ID NO:2, comprising: a) contacting apolypeptide of claim 21 with a test compound; and b) determining theeffect of the test compound on the activity of the polypeptide tothereby identify a compound which modulates the activity of thepolypeptide.